μM Cu2 Research Articles

Extracellular Acidosis, Cysteine, and Glutathione Enhance the Toxic Effect of Copper Ions in Cultures of Cerebellar Granule Neurons.

We studied the effect of extracellular acidosis, cysteine, glutathione, and iron ions (Fe3+) on the neurocytotoxic effect of copper ions (Cu2+) in vitro. At acidic pH of the culture medium (pH 6.8), the toxic effect of copper on cultured neurons significantly increased in comparison with that at neutral pH 7.3. In the presence of 25 μM Cu2+ in the culture medium at pH 7.3 and 6.8, the neuronal survival was 89±2 and 63±4%, respectively. In the presence of glutathione or cysteine (1 μM) in the culture medium, even 0.5 μM Cu2+ caused 100% death of cultured neurons, while the presence of Fe3+ (10-50 μM) had no effect on the toxicity of Cu2+. In general, acidosis or the presence of glutathione or cysteine increases the cytotoxicity of copper ions.

Characteristics of the Fatty Acid Composition of the Vacuolar Membrane Lipids Under the Conditions of Stress Induced by Copper Ions

The effect of different concentrations of copper ions (100 and 500 µM) on the fatty acid composition of the vacuolar membrane lipids of beet root tissues (Beta vulgaris L.) was studied. Exposure to 100 μM copper led to an increase in the content of FAs by 57 μg/mg of total lipids as compared to the control. Stearoyl desaturase (SDR) activity decreased from 0.87 in the control to 0.77 at 100 μM copper. Exposure to 500 μM copper caused an increase in the SDR activity up to 0.93, but the proportion of FAs decreased by 50 μg/mg of total lipids compared with 100 μM copper. In addition, there was an increase in the saturation of tonoplast lipids to 44 and 40% at 100 and 500 μM Cu²⁺, respectively. The results suggest that FAs of tonoplast lipids are involved in the stress response mechanisms induced by excessive Cu²⁺ concentrations.

Effects of copper, nickel and lead on growth parameters and antioxidative defense system of Solanum lycopersicum L.

The current study assessed the effects of lead (Pb), copper (Cu), and nickel (Ni) in roots and shoots on growth indices, the antioxidative defense system, and metal uptake in Solanum lycopersicum L. variety Punjab Kesar Cherry. For 60 days, S. lycopersicum seeds were exposed to varying amounts of three metals (0-100 μM of Cu and 0-60 μM of Ni and Pb). In comparison to the control, the percentage of germination, root and shoot length, and fresh and dry weight of the roots and shoots all decreased, according to the results. The bioaccumulation factor of both roots and shoots, along with the translocation factor, increased at lower concentrations and decreased at higher concentrations; for Pb, on the other hand, the translocation factor increased with increasing concentrations. At 60 μM, the order of the bioaccumulation factor was Cu>Ni>Pb for roots, and Cu>Pb>Ni for shoots. The antioxidative enzyme activities, including ascorbate peroxidase (APX), catalase (CAT), dehydro ascorbate reductase (DHAR), glutathione reductase (GR), glutathione S transferase (GST), peroxidase (POD), and superoxide dismutase (SOD), were increased at lower concentrations and decreased at higher concentrations under Cu, Ni, and Pb treatments. The order of toxicity in terms of decrease in protein content was observed as Pb>Ni>Cu for both roots and shoots.

Effects of Ferulic Acid on Respiratory Metabolism, Oxidative Lesions, and Apoptotic Parameters in Gills and Red Blood Cells of Carp (Cyprinus carpio Var. Jian) Response to Copper.

In sustainable aquaculture systems, copper sulfate (CuSO4) is widely applied as a disinfectant to control parasitic infections and algal growth. However, aquatic organisms may suffer from exposure to excessive concentrations of Cu. Elevated Cu concentrations could activate damage to the respiratory functions of aquatic animals. Thus, this study explored the effects exerted by ferulic acid (FA) on respiratory metabolism, oxidation-related lesions, and the apoptosis parameters of the gills and red blood cells in copper sulfate pentahydrate (CuSO4·5H2O)-treated carp (Cyprinus carpio var. Jian). When the 30-day feeding experiment was completed, the carp were exposed to 12.5 μM of Cu for 4 days. The results indicated that the Cu decreased the oxygen consumption and ammonia excretion rates in the carp, reduced the metabolic- and antioxidant-related enzyme activities and glutathione levels in the carp, and enhanced the caspase activities and reactive oxygen species and malondialdehyde levels in the gills of the carp. Moreover, in addition to the changes in the above parameters, the Cu decreased the cell numbers and hemoglobin concentrations and increased the phosphatidylserine exposure and cytochrome c levels in the red blood cells of the carp. These results demonstrate that Cu is capable of decreasing respiratory metabolism and increasing oxidation-related lesions and apoptosis inside the gills and red blood cells of the fish. However, dietary FA quenched the Cu-induced apoptosis and oxidative lesions by reversing the same biomarker indicators, thereby suppressing the Cu-induced decrease in respiratory metabolism. Thus, FA can be used as a suppressor of Cu stress in fish.

Copper Ions Reduced Toxicity of Sodium Azide and Lipopolysaccharide on Cultured Cerebellar Granule Neurons

Introduction. Copper ions (Cu2+) are structural elements of proteins such as cytochrome с oxidase (Complex IV), an enzyme that catalyzes the final step of electron transfer to oxygen during oxidative phosphorylation in the mitochondria. With Cu2+ homeostasis being of utmost importance, its disturbances in the central nervous system are involved in the mechanisms of many neurodegenerative and other brain disorders.
 This study aimed to assess the effects of non-toxic copper ion levels on death of cerebellar granule neurons associated with lipopolysaccharide (LPS; in vitro inflammation model) or azide sodium (NaN3; cytochrome с oxidase inhibitor).
 Materials and methods. LPS (10 μg/mL) or NaN3 (250 μM) was added on day 7 to 8 to the culture medium with rat cerebellar cells for 24 hours in vitro. Nitrite concentrations were measured in the culture medium by Griess assay; absorbance was recorded with a spectrophotometer at 540 nm, and morphologically intact cells were counted as survived neurons.
 Results. Added to the culture medium, LPS or NaN3 reduced neuron survival to 15 ± 2% or 20 ± 3% vs. control, respectively. Cu2+ (0.5 to 5.0 μM) increased neuron survival in a dose-dependent manner to 78 ± 4% with toxic levels of LPS and to 86 ± 6% with NaN3 with 5 μM Cu2+. The concentration of nitrites in the control culture medium was 2.0 ± 0.2 μM. Added to the cell cultures, LPS increased the concentration of nitrites to 8.5 ± 0.5 μM. Cu2+ 5 μM did not show any significant effects on nitrite accumulation in the culture medium.
 Conclusions. We showed that copper ions can exert protective effects on neurons against LPS-induced or NaN3-induced toxicity. This protection is likely to be associated rather with Cu2+ interaction with Complex IV of the electron transfer chain in the mitochondria than with inhibition of NO production. Effects of Cu2+ on apoptosis pathway proteins also cannot be ruled out.

Copper-Induced InVivo Gene Amplification in Budding Yeast.

In the biotechnological industry, multicopy gene integration represents an effective strategy to maintain a high-level production of recombinant proteins and to assemble multigene biochemical pathways. In this study, we developed copper-induced invivo gene amplification in budding yeast for multicopy gene expressions. To make copper as an effective selection pressure, we first constructed a copper-sensitive yeast strain by deleting the CUP1 gene encoding a small metallothionein-like protein for copper resistance. Subsequently, the reporter gene fused with a proline-glutamate-serine-threonine-destabilized CUP1 was integrated at the δ sites of retrotransposon (Ty) elements to counter the copper toxicity at 100 μM Cu2+. We further demonstrated the feasibility of modulating chromosomal rearrangements for increased protein expression under higher copper concentrations. In addition, we also demonstrated a simplified design of integrating the expression cassette at the CUP1 locus to achieve tandem duplication under high concentrations of copper. Taken together, we envision that this method of copper-induced invivo gene amplification would serve as a robust and useful method for protein overproduction and metabolic engineering applications in budding yeast.

Protective role of fullerenol and arginine C60 fullerene against copper toxicity in cucumber

Copper (Cu), when in excess, is one of the most toxic and hazardous metals to all living organisms, including plants. Engineered nanomaterials have the potential for increasing crop protection. However, the protective role of fullerenes (carbon-based nanoparticles with wide application in various areas) against Cu toxicity in plants is, so far, understudied. The present study investigated whether fullerenes can potentially alleviate Cu toxicity in plants (Cucumis sativus L.). Two water-soluble fullerene C60 derivatives were examined: fullerenol [C60(OH)22–24] and arginine-functionalized fullerene [C60(C6H13N4O2)8H8], under controlled conditions using hydroponics. Plants treated with 15 μM of Cu exhibited typical symptoms of Cu toxicity: impaired growth, leaf chlorosis, reduced photosynthetic activity, nutritional imbalances, and enhanced lipid peroxidation. These symptoms were alleviated in the presence of fullerene derivatives with arginine C60 having the more pronounced effect. Improved cucumber Cu tolerance was attributable to Cu buffering in the root zone (roots and medium), which caused a dramatic decline in Cu transport towards leaves and the elimination of oxidative damage. The Cu removal efficacy of arginine C60 was much greater than that of fullerenol. These fullerenes acted in a dose-dependent manner and removed Cu selectively without significant modification of the bioavailability of other essential nutrients. Treatment with free arginine did not affect Cu immobilization or Cu toxicity. These results suggest that the surface chemistry of the fullerene core is important for the protection of plants under excessive Cu conditions. The information offered a new approach to preparing promising practical materials for alleviating Cu toxicity in plants with potential application in fields.

Assessment of Gus Expression Induced by Anti-Sense OsPPO Gene Promoter and Antioxidant Enzymatic Assays in Response to Drought and Heavy Metal Stress in Transgenic Arabidopsis thaliana

Abiotic stresses, including drought and heavy metals, are detrimental to plant growth and development and enormously reduce agricultural yields. Plants may quickly change their transcriptome in response to various stressful conditions. Plants develop many defense mechanisms to respond to various stresses that can be classified into morphological, physiological, and biochemical responses. Polyphenol oxidases (PPOs) are one of the self-protective enzymes found in plants except for Arabidopsis. Currently, drought and heavy metals were applied exogenously to transgenic A. thaliana lines (transformed with Oryza sativa PPO promoter fused to the GUS reporter gene). The current study mainly focused on the systematic pathway by which plants respond to stressors. The aim of this study is to investigate the effect/expression of PPO and antioxidant defense system against abiotic stresses. A. thaliana was treated with different concentrations of polyethylene glycols. At 30% PEG, maximum fold induction (1.9) was seen after 12 h. Overall, various concentrations (5%, 20%, and 30%) induced PPO expression after 6, 12, and 24 h. Moreover, three different concentrations of Cu (50 µM, 100 µM, 200 µM) and Ni (50 µM, 100 µM, 200 µM) for 6, 12, and 24 h were also applied. It was observed that the expression profiling of the OsPPO promoter induced GUS gene expression in response to Cu and Ni treatments. The maximum fold induction (15.03) of GUS was observed in 100 µM of Cu after 24 h. In the case of Ni, maximum fold induction of (7.78) was observed at 100 µM after 24 h. So, both Cu and Ni showed a similar pattern of induction at 100 µM after 24 h. In conclusion, the efficiency of the PPOGUS promoter can be operated to assess the response of plants to various abiotic stimuli.

Highly sensitive fiber optic sensor based on chitosan/α-Fe2O3 for trace copper(II) ion detection

Instrument methods are essential for simple and rapid detection of copper(II) (Cu2+) in drinking water and healthcare. However, these methods suffer from complex sample pretreatment and long detection time. This paper proposed a highly sensitive fiber optic sensor for trace Cu2+ detection in aqueous solutions. Based on the principle of modal interference, by surface functionalization, a uniform chitosan (CS)/α-Fe2O3 nanocomposite material was coated on a structure of no-core fiber–multimode fiber–no-core fiber (NCF1-MMF-NCF2). The amino and hydroxyl groups in the material have a specific recognition function for Cu2+, which can chelate with Cu2+ and change the refractive index of the material, leading to the shift of interference spectrum. Trace Cu2+ detection can be achieved by monitoring the displacement of the interference spectrum. The results revealed that within the range of 0–1 μM Cu2+ concentrations, with increasing Cu2+ concentration, the interference spectrum underwent a red shift and the maximum detection sensitivity of Cu2+ ions reached 456.69 nm/μM, with a good linearity of 0.989. Also, the sensor has the advantages of high stability, good repeatability, simple fabrication, and low cost.

Cu2+-dependent hydrolysis of O-hexyl 2,5-dichlorophenyl phosphoramidate by reptile sera

This study shows the EDTA-resistant, Ca2+ and Cu2+-dependent hydrolysis of O-hexyl 2,5-dichlorophenyl phosphoramidate (HDCP) compound in reptiles sera determined by spectrophotometry UV/Vis and chiral chromatography. Samples of ten reptile species were incubated with aliquot of 100 or 400 μM HDCP in presence of 100 or 300 μM Cu2+, or 2.5 mM Ca2+ or 5 mM EDTA at 37 °C for 30–60 min. The results shown an activator effect of Cu2+ on HDCP hydrolysis in freshwater turtles sera (Trachemys scripta, Chelydra serpentina and Macrochelys temminckii) because the levels of 2,5-dichlorophenol (DCP; product hydrolysis) were similar (∼37 μM DCP) to chicken serum (positive control group). The marine turtles (Chelonia mydas and Eretmochelys imbricata) and crocodiles (Crocodylusacutus and Crocodylus moreletii) showed ∼50% less HDCPase activity (13–17 μM DCP) compared to the HDCPase activity of the freshwater turtle species. Terrestrial reptile species (snakes and lizards) showed around 25% of activity (7–13 μM DCP) with both copper concentrations. These Cu2+-dependent hydrolysis were stereospecific to R(+)-HDCP (p˂0.05) in the three freshwater turtle species that showed similar hydrolysis to the chicken serum. However, the Ca2+ did not show a significant activating effect on the HDCPase activity (1–8 μM DCP) in any reptile serum. Their hydrolysis levels were very similar to those of EDTA-resistant activity. The present study demonstrates a Cu2+-dependent A-esterase (HDCPase) activity in turtles and points serum albumin as the cuproprotein responsible for this activity, reinforcing its N-terminal sequence (DAEH) as a catalytic center.

Europium metal-organic frameworks: Synthesis, characterization, and application as fluorescence sensors for the detection of Cu2+, Ni2+ cations and T3, T4 hormones

The solvothermal approach was used to create a novel Eu metal-organic framework (Eu-MOF) based on 1,4-benzendicarboxylic acid (TPA), 1,10-phenanthroline, and N,N-dimethylformamide (DMF)/H2O. Structural analysis of Eu-MOF, Fluorescence spectrometry, Fourier Transform Infrared Spectrometer (FTIR), Scanning Electron Microscopy (SEM), Energy dispersive X-ray (EDX) mapping, Thermo-gravimetric analysis (TGA), and Single Crystal X-Ray Diffraction (PXRD) methods. Using the fluorescence properties of the synthesized Eu-MOF, its use as a fluorescence sensor in the determination of different analytes, such as organic molecules (T3-T4 hormone, ascorbic acid, and glucose) and metal ions (Na+, K+, Ca2+, Mg2+, Cu2+, Mn2+, Hg2+, Pb2+, Ni2+, Cr3+, Al3+, Fe3+), was investigated. Fluorescence experiments revealed that Cu2+, Ni2+ cations, as well as T3 and T4 hormones, quenched the fluorescence of Eu-MOF. Turn-off luminescence can be induced by 10 μM Cu2+, 30 μM Ni2+ cations, 500 nM T3, and 800 nM T4 hormones. Fluorescence quenching efficiencies were calculated for Cu2+, Ni2+, T3, and T4 99.7%, 99.6%, 98.7%, and 98.2%, respectively.

Expression of a deep-sea bacterial laccase from Halomonas alkaliantartica and its application in dyes decolorization

IntroductionLaccase is a copper-containing polyphenolic oxidase widely found in bacteria, archaea, fungi, animals, and plants. As a green biocatalyst with considerable potential for numerous environmental and industrial applications, the enzyme production efficiency of laccase in nature is low, and the cost is high.PurposeTo examine the characterization and potential applications of laccase in this study, a novel laccase from Halomonas alkaliantartica (LacHa) was cloned and heterologously expressed it in Escherichia coli.ResultsTo achieve heterologous and efficient laccase expression, a bacterial laccase gene designed as LacHa from Halomonas alkaliantartica of deep sea was cloned and expressed in E. coli. The results showed that the optimum temperature and pH of the enzyme reaction were 45 °C and 7.5. The 100 μM Cu2+ and Fe2+ ions had the strongest stimulatory effect on laccase activity, the surface-active agent SDS and organic solvent 5% ethanol had opposite effect. EDTA, and 5% DMSO have no effect on LacHa activity.The activity of LacHa was enhanced 1.5-fold by chloride at concentrations lower than 500 mM, and 57.6% of its initial activity remained in the reaction system containing 1000 mM.NaCl. Furthermore, LacHa showed decolorization rates ranging from 90.28 to 100% for indigo carmine and two azo dyes without mediators, with wide pH (5.0–9.0) and temperature (25–65 °C) ranges.ConclusionsIn this study, LacHa was expressed and showed unusual properties, indicating its great application potential in textile industries or environmental fields.

In vitro and in vivo evaluation of diethyldithiocarbamate with copper ions and its liposomal formulation for the treatment of Staphylococcus aureus and Staphylococcus epidermidis biofilms

Surgical site infections (SSIs) are mainly caused by Staphylococcus aureus (S. aureus) and Staphylococcus epidermidis (S. epidermidis) biofilms. Biofilms are aggregates of bacteria embedded in a self-produced matrix that offers protection against antibiotics and promotes the spread of antibiotic-resistance in bacteria. Consequently, antibiotic treatment frequently fails, resulting in the need for alternative therapies. The present study describes the in vitro efficacy of the Cu(DDC)2 complex (2:1 M ratio of diethyldithiocarbamate (DDC−) and Cu2+) with additional Cu2+ against S. aureus and S. epidermidis biofilms in models mimicking SSIs and in vitro antibacterial activity of a liposomal Cu(DDC)2 + Cu2+ formulation. The in vitro activity on S. aureus and S. epidermidis biofilms grown on two hernia mesh materials and in a wound model was determined by colony forming unit (CFU) counting. Cu2+-liposomes and Cu(DDC)2-liposomes were prepared, and their antibacterial activity was assessed in vitro using the alamarBlue assay and CFU counting and in vivo using a Galleria mellonella infection model. The combination of 35 μM DDC− and 128 μM Cu2+ inhibited S. aureus and S. epidermidis biofilms on meshes and in a wound infection model. Cu(DDC)2-liposomes + free Cu2+ displayed similar antibiofilm activity to free Cu(DDC)2 + Cu2+, and significantly increased the survival of S. epidermidis-infected larvae. Whilst Cu(DDC)2 + Cu2+ showed substantial antibiofilm activity in vitro against clinically relevant biofilms, its application in mammalian in vivo models is limited by solubility. The liposomal Cu(DDC)2 + Cu2+ formulation showed antibiofilm activity in vitro and antibacterial activity and low toxicity in G. mellonella, making it a suitable water-soluble formulation for future application on infected wounds in animal trials.

Applying synchronous fluorescence spectroscopy combined with Gaussian band fitting and two-dimensional correlation to characterize interactions of copper (II) with dissolved organic matter from urban river sediments

Dissolved organic matter (DOM) and heavy metals can form strong complexes, which might determine distribution, toxicity, bioavailability, and environmental trend of heavy metals in aquatic environment. Synchronous fluorescence spectroscopy combined with Gaussian band fitting and two-dimensional correlation (2DCOS) was applied to characterize Cu2+ interactions with DOM fractions from sediments in Xihe River. DOM from four layers was mainly associated with tyrosine-like (TYLF), tryptophan-like (TRLF), microbial humic-like (MHLF), fulvic-like (FLF), and humic-like fluorescence (HLF) substances based on Gaussian band fitting. At the first layer, the f value of FLF was the highest among five components, while TRLF at the second and third layers represented the highest f values. Besides, at the fourth layer, the f value of TYLF was the highest. According to 2DCOS and moving window 2DCOS, the decreasing binding sensitivities were HLF→MHLF→TYLF→TRLF→FLF in the first layer, which almost represented high efficiencies at 40 μM Cu2+. The binding order was MHLF→HLF→FLF→TRLF→TYLF in the second layer, where TYLF and TRLF shared the identical efficiencies at 120 μM Cu2+. For the third layer, the varying order was TYLF→FLF→TRLF→MHLF→HLF, whose high efficiencies occurred at 10 μM Cu2+. In the fourth layer, the order was TYLF→FLF→MHLF→TRLF→HLF, whose efficiencies almost covered whole titrate concentrations. At a given layer, structural equation model showed Cu2+ concentration negatively affected the fluorescence intensity of DOM components except FLF at the substrate layer. This study might provide an effective method to illustrate the interaction mechanism of the heavy metals and DOM fractions.

Metabolomic analysis reveals the molecular responses to copper toxicity in rice (Oryza sativa)

Copper (Cu) is one of the essential microelements and widely participates in various pathways in plants, but excess Cu in plant cells could induce oxidative stress and harm plant growth. Rice (Oryza sativa) is a main crop food worldwide. The molecular mechanisms of rice in response to copper toxicity are still not well understood. In this study, two-week-old seedlings of the rice cultivar Nipponbare were treated with 100 μM Cu2+ (CuSO4) in the external solution for 10 days. Physiological analysis showed that excess Cu significantly inhibited the growth and biomass of rice seedlings. After Cu treatment, the contents of Mn and Zn were significantly reduced in the roots and shoots, while the Fe content was significantly increased in the roots. Meanwhile, the activities of antioxidant enzymes including SOD and POD were dramatically enhanced after Cu treatment. Based on metabolomic analysis using liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods, 695 metabolites were identified in rice roots. Among these metabolites, 123 metabolites were up-regulated and 297 were down-regulated, respectively. The differential metabolites (DMs) include carboxylic acids and derivatives, benzene and substituted derivatives, carbonyl compounds, cinnamic acids and derivatives, fatty acyls and organ nitrogen compounds. KEGG analysis showed that these DMs were mainly enriched in TCA cycle, purine metabolism and starch and sucrose metabolism pathways. Many intermediates in the TCA cycle and purine metabolism were down-regulated, indicating a perturbed carbohydrate and nucleic acid metabolism. Taken together, the present study provides new insights into the mechanism of rice roots to Cu toxicity.

Copper, cadmium and nickel pollution inhibit growth and promote ascorbate catabolism in cell cultures of Arabidopsis thaliana and Zea mays

We established model systems for exploring the roles of symplastic and apoplastic ascorbate in heavy-metal-polluted dicot and monocot cells. Cell-suspension cultures of Arabidopsis and maize were treated with copper, cadmium or nickel; growth and ascorbate metabolism were measured. Growth was halved by ∼80 µM Cu2+, 90 µM Cd2+ or 1200 µM Ni2+ in Arabidopsis, and ∼90 µM Cu2+, 650 µM Cd2+ or 650 µM Ni2+ in maize. Cu2+ (128 µM) and Cd2+ (512 µM) caused partial loss of symplastic ascorbate, especially in Arabidopsis; Ni2+ (512 and 2048 µM) had moderate effects. Added apoplastic l-ascorbate (1 mM) was consumed by the cultures (half-life ∼23 and 44 min in Arabidopsis and maize, respectively), consumption rate being 3–6-fold increased by Cu2+, Cd2+ and Ni2+ in Arabidopsis, and by Cu2+ in maize; Cd2+ and Ni2+ had relatively little effect on apoplastic ascorbate consumption in maize. Radioactivity from exogenous 1 mM l-[1-14C]ascorbate remained extracellular; catabolites formed were dehydroascorbic acid, diketogulonate and oxalyl-threonates. In conclusion, suspension-cultured cells respond to heavy-metal stresses by maintaining symplastic ascorbate concentrations, which may beneficially scavenge symplastic reactive oxygen species (ROS). Apoplastic ascorbate is catabolised in metal-polluted cultures via several oxidative and non-oxidative reactions, the former potentially scavenging stress-related apoplastic ROS.

Long-term waterborne Cu2+ exposure affects collagen metabolism in fish

Copper pollution might have a negative effect on collagen metabolism in fish. To test this hypothesis, we exposed an important economical fish, silver pomfret (Pampus argenteus), to three concentrations of Cu2+ for up to 21 days to simulate natural exposure to copper. With increasing copper exposure concentration and time, hematoxylin and eosin staining and picrosirius red staining revealed extensive vacuolization, cell necrosis, and tissue structure destruction, and a change of type and abnormal accumulation of collagen in the liver, intestine, and muscle tissues. To further study the mechanism of collagen metabolism disorder caused by copper exposure, we cloned and analyzed a key collagen metabolism regulation gene, timp, of silver pomfret. The full-length timp2b cDNA was 1035 bp with an open reading frame of 663 bp, encoding a protein of 220 amino acids. Copper treatment significantly increased the expression of akts, erks, and fgfs genes and decreased the mRNA and protein expression of Timp2b and MMPs. Finally, we constructed a silver pomfret muscle cell line (PaM) for the first time and used PaM Cu2+ exposure models (450 μM Cu2+ exposure for 9 h) to examine regulation function of the timp2b-mmps system. We knocked down or overexpressed timp2b in the model, and found that downregulation of mmps expression and upregulation of akt/erk/fgf were further aggravated in the timp2b− group (subjected to RNA interference), whereas some recovery was achieved in the timp2b+ group (overexpression). These results indicated that long-term excessive copper exposure can lead to tissue damage and abnormal collagen metabolism in fish, which might be caused by the alteration of akt/erk/fgf expression, which disrupts the effects of the timp2b-mmps system on extracellular matrix balance. The present study assessed the impact of copper on the collagen of fish and clarified its regulatory mechanism, providing a basis for toxicity of copper pollution study.

A high-performance fluorescent and ratiometric colorimetric detection of Cu2+ in practice.

To monitor Cu2+ efficiently, a kind of D-π-A-π-D conjugated 3,5-di-(2-hydroxyl naphthaldehyde)-iminyl triazole (HNIT) was developed, using triazole as the electron acceptor, 2-hydroxyl naphthaline as the electron donor, and -CN- as the bridging group. The proposed HNIT possessed superior UV-vis and fluorescent spectral property with high molar absorption coefficient of 2.313 × 104 L mol-1 cm-1 and fluorescence quantum yield of 36.2%. Trace Cu2+ could exclusively alter its UV-vis and fluorescent property with clear color change. Under the optimized conditions, a high-performance fluorescent and ratiometric colorimetric detection of Cu2+ based on HNIT was efficient, with low detection limits of 3.3 × 10-8 mol L-1 (S/N = 3) and 9.6 × 10-8 mol L-1 (S/N = 3), respectively. It well satisfied with the safe value of 31.5 μM Cu2+ in drinking water recommended by World Health Organization (WHO). When applied for detection of Cu2+ in real environmental samples, the recovery was in the range of 97.5-105.2%. The recognition mechanism for HNIT to Cu2+ realized quite stable 6-membered rings between electron-deficient Cu2+ and electron-rich N and O atoms in HNIT with 1 : 2 chemical stoichiometry of HNIT to Cu2+.

A Novel Fluorescent Chemodosimeter for Discriminative Detection of Cu2+ in Environmental, Food, and Biological Samples

Copper-ion pollution has serious harm to the environment and human health, and abnormal doses of Cu2+ can cause a series of diseases. The technology for the detection of Cu2+ has been constantly updated and developed. Food is the main source of Cu2+ in the human body, but the current detection methods of Cu2+ still have a variety of limitations such as complex operational processes, time consumption, and so on. Therefore, in this work, a new type of Cu2+ fluorescent chemodosimeter GT-TSC was developed. With regard to the structure of GT-TSC, 4-CF3-7-aminoquinoline was selected as the fluorescent group of GT-TSC, and thiosemicarbazone was used as a new recognition receptor for Cu2+, which can be complexed with Cu2+. The fluorescence intensity of GT-TSC had an excellent linear relationship with 0.1–10 μM Cu2+. The detection limit of GT-TSC was 0.065 μM, which means that GT-TSC had a rapid and accurate response ability to Cu2+ in the samples. Meanwhile, GT-TSC had good Golgi localization ability. Importantly, GT-TSC has been successful in the detection of Cu2+ in the environmental, food, and even biological samples. Obviously, it is conceivable that GT-TSC can be applied to more practical samples and develop into a potential tool for detecting Cu2+.

Effect of nano-silicon on the regulation of ascorbate-glutathione contents, antioxidant defense system and growth of copper stressed wheat (Triticum aestivum L.) seedlings.

Copper (Cu2+) toxicity can inhibit plant growth and development. It has been shown that silicon (Si) can relieve Cu2+ stress. However, it is unclear how Si-nanoparticles (SiNPs) relieve Cu2+ stress in wheat seedlings. Therefore, the current study was conducted by setting up four treatments: CK, SiNP: (2.5 mM), Cu2+: (500 µM), and SiNP+Cu2+: (2.5 mM SiNP+500 µM Cu2+) to explore whether SiNPs can alleviate Cu2+ toxicity in wheat seedlings. The results showed that Cu2+ stress hampered root and shoot growth and accumulated high Cu2+ concentrations in roots (45.35 mg/kg) and shoots (25.70 mg/kg) of wheat as compared to control treatment. Moreover, Cu2+ treatment inhibited photosynthetic traits and chlorophyll contents as well as disturbed the antioxidant defense system by accumulating malondialdehyde (MDA) and hydrogen peroxidase (H2O2) contents. However, SiNPs treatment increased root length and shoot height by 15.1% and 22%, respectively, under Cu2+ toxicity. Moreover, SiNPs application decreased MDA and H2O2 contents by 31.25% and 19.25%, respectively. SiNPs increased non-enzymatic compounds such as ascorbic acid-glutathione (AsA-GSH) and enhanced superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and ascorbic peroxidase (APX) activities by 77.5%, 141.7%, 68%, and 80%, respectively. Furthermore, SiNPs decreased Cu2+ concentrations in shoots by 26.2%, as compared to Cu2+ treatment alone. The results concluded that SiNPs could alleviate Cu2+ stress in wheat seedlings. The present investigation may help to increase wheat production in Cu2+ contaminated soils.