Toxicity of common biocides used in aquaculture to embryos and larvae of Brachymystax tsinlingensis Li.

Brachymystax tsinlingensis Li is a threatened fish species endemic to China. With the problems of environmental factors and seeding breeding diseases, it is important to further improve the efficiency of seeding breeding and the basis of resource protection. This study investigated the acute toxicity of copper, zinc and methylene blue (MB) on hatching, survival, morphology, heart rate (HR) and stress behaviour of B. tsinlingensis. Eggs (diameter: 3.86 ± 0.07 mm, weight: 0.032 ± 0.004 g) of B. tsinlingensis were selected randomly from artificial propagation and developed from eye-pigmentation-stage embryos to yolk-sac stage larvae (length: 12.40 ± 0.02 mm, weight: 0.03 ± 0.001 g) and exposed to different concentrations of Cu, Zn and MB for 144 h in a series of semi-static toxicity tests. The acute toxicity tests indicated that the 96-h median lethal concentration (LC50 ) values of the embryos and larvae were 1.71 and 0.22 mg l-1 for copper and 2.57 and 2.72 mg l-1 for zinc, respectively, whereas the MB LC50 after 144-h exposure for embryos and larvae were 67.88 and 17.81 mg l-1 , respectively. The safe concentrations of copper, zinc and MB were 0.17, 0.77 and 6.79 mg l-1 for embryos and 0.03, 0.03 and 1.78 mg l-1 for larvae, respectively. Copper, zinc and MB treatments with concentrations greater than 1.60, 2.00 and 60.00 mg l-1 , respectively, led to a significantly low hatching rate and significantly high embryo mortality (P < 0.05), and copper and MB treatments with concentrations greater than 0.2 and 20 mg l-1 led to significantly high larvae mortality (P < 0.05). Exposure to copper, zinc and MB resulted in developmental defects, including spinal curvature, tail deformity, vascular system anomalies and discolouration. Moreover, copper exposure significantly reduced the HR of larvae (P < 0.05). The embryos exhibited an obvious change in behaviour, converting from the normal behaviour of emerging from the membrane head first to emerging tail first, with probabilities of 34.82%, 14.81% and 49.07% under copper, zinc and MB treatments, respectively. The results demonstrated that the sensitivity of yolk-sac larvae to copper and MB was significantly higher than that of embryos (P < 0.05) and that B. tsinlingensis embryos or larvae might be more resistant to copper, zinc and MB than other members of the Salmonidae family, which benefits their resource protection and restoration.

Triazophos exposure on maternal Daphnia magna at environmental-related concentrations revealed toxic effects to its offspring.

Due to chemical and photochemical stability, triazophos has been frequently detected in rivers and oceans over the years with extensive use for pest control in agriculture, and it has become a worldwide ecological concern to the aquatic environment. Until now, fewer data are available regarding the potential long-term adverse effects of triazophos on aquatic invertebrates, which plays an essential role in aquatic food webs, as a key group for water ecosystems. In this experiment, the F1- and F2 progenies of Daphnia magna were recovered when daphnias (F0) exposure to triazophos at environmental-related concentrations (0.1 and 1.0 µg/L) for 21 d; and the indexes related to phenotypic traits, reproduction and gene expression were measured in tested animals. The results showed that heart rate and total number of neonates in exposed F0-daphnias were significantly lower than those of control group, and the detoxification genes (HR96 and P-gp) were up-regulated while genes related reproduction (Vtg) and molting (Nvd and Shd) were significantly down-regulated. The heart rate and individual size of F1-daphnias (<24 h) were significantly reduced in the treatment group. After 21-d recovery, the heart rate and expression of HR96, P-gp, Vtg, Nvd and Shd were declined in F1-daphnias. There was no obvious difference of morphological traits and heart rate between treatment and control in F2-daphnias (<24 h). In summary, daphnias (F0) exposure to triazophos with environmental dose could raise toxic effects on its offspring (F1), which is mainly manifested by reduced heart rate, the accumulated number and individual size of offspring and decreased expression of genes related to molting and reproduction.

Molecular cloning and functional characterization of melanocortin-3 receptor in grass carp (Ctenopharyngodon idella).

The melanocortin-3-receptor (MC3R) plays an important role in mammals' food intake and energy homeostasis. However, its physiological role in bony fishes, such as grass carp, has not been well understood. This study reports the molecular cloning, tissue distribution, and pharmacological characterization of grass carp melanocortin-3-receptor (ciMC3R). Phylogenetic and chromosomal synteny analyses indicated that ciMC3R was closest to cyprinid fishes in evolution. Quantitative PCR experiments revealed that the mRNA of ciMC3R was highly expressed in the brain of grass carp. The cytological function of ciMC3R was investigated by the co-transfection of pcDNA3.1-ciMC3R and the signal-pathway-specific luciferase into the HEK293T cells. Results revealed that the four agonists, α-MSH, ß-MSH, ACTH, and NDP-MSH, potentiate the activation of ciMC3R and further increase the production of cAMP and upregulate the MAPK/ERK signaling, respectively. Our study will provide basic data for exploring the physiological functions of grass carp MC3R, especially in energy homeostasis and food intake.

The interaction of MC3R and MC4R with MRAP2a in rainbow trout (Oncorhynchus mykiss).

Melanocortin 3 and 4 receptors are two important neural G protein-coupled receptors that regulate energy homeostasis in vertebrates. Melanocortin receptor accessory protein 2 (MRAP2) is also involved in the regulation of food intake and body weight as a variable regulator of melanocortin receptors. Rainbow trout (Oncorhynchus mykiss) is a valuable cold-water fish cultured worldwide. In the rainbow trout model, we cloned and identified mrap2a, a paralog of mrap2. Rainbow trout mrap2a consisted of a 690 bp ORF and was expected to encode a putative protein of 229 amino acids. The qPCR results showed that rainbow trout mrap2a was expressed at high levels in brain tissue similar to mc3r and mc4r. In addition, co-immunoprecipitation verified that MRAP2a interacts with MC3R and MC4R in vitro and that MRAP2a is involved in and regulates the constitutive activity and signaling of MC3R and MC4R. MRAP2a reduced constitutive and agonist-stimulated cAMP levels of MC3R; furthermore, MRAP2a increased constitutive ERK1/2 activation but reduced ligand-induced stimulation at high levels of expression. For MC4R, MRAP2a showed decreased cAMP basal activity but increased agonist-stimulated cAMP signaling and increased ACTH ligand sensitivity. However, MRAP2a failed to affect MC4R constitutive activity and agonist-induced ERK1/2 signaling. Undoubtedly, our study will have great significance for revealing the conserved role of MC4R and MC3R signaling in teleost fish, especially in cold-water fish growth and energy homeostasis.

Fabrication of avidin-stabilized gold nanoclusters with dual emissions and their application in biosensing.

Protein-stabilized gold nanoclusters (Prot-Au NCs) have been widely used in biosensing and cell imaging owing to their excellent optical properties and low biotoxicity. However, several Prot-Au NCs reported in the literature do not retain the biological role of the protein, which greatly limits their ability to directly detect biomarkers. This study demonstrated for the first time the successful synthesis of dual-function avidin-stabilized gold nanoclusters (Av-Au NCs) using a one-pot method. The resulting Av-Au NCs exhibited intense blue and red emissions under 374 nm excitation. Furthermore, the Av-Au NCs retained the native functionality of avidin to bind to biotin. When DNA strands modified with biotin at both ends (i.e., linker chains) were mixed with Av-Au NCs, large polymers were formed, indicating that Av-Au NCs could achieve fluorescence signal amplification by interacting with biotin. Taking advantage of the aforementioned properties, we constructed a novel enzyme-free fluorescent biosensor based on the Av-Au NCs-biotin system to detect DNA. The designed fluorescent biosensor could detect target DNA down to 0.043 nM, with a wide line range from 0.2 nM to 20 µM. Thus, these dual-functional Av-Au NCs were shown to be an excellent fluorescent material for biosensing.

Evaluation of the acute toxic response induced by triazophos to the non-target green algae Chlorella pyrenoidosa.

Residues of triazophos in aquatic ecosystems due to extensive use for controlling pests in agriculture has became worldwide concern, while the toxic response of triazophos on the non-target green algae in aquatic environment is not well studied. Therefore, the acute (96 h) toxic effects of 1 and 10 mg/L triazophos on green algae Chlorella pyrenoidosa were evaluated in present study. The results showed that the growth was notably inhibited when treated with triazophos and the 96 h-EC50 (median inhibition concentration) were 12.79 mg/L. The content of photosynthetic pigments (including chl a, chl b, total-chl and carotinoids) clearly decreased under two treatments after 48 h and 96 h with exception for the values at 48 h exposure in 1 mg/L treatment. In addition, the transcript abundance of photosynthesis-related genes (psbA, psbC and rbcL) showed obvious decrease in above two treatments after exposure 96 h to triazophos. In response to 10 mg/L triazophos treatment, the morphology of thylakoid chloroplast of algal cells were obviously damaged. It was also found that starch granules increased with down-regulation of atpB gene expression in 10 mg/L treatment, which suggests that triazophos may inhibit the energy metabolism of C. pyrenoidosa. Moreover, the algal growth inhibition was along with the increase of intracellular reactive oxygen species (ROS), activity of antioxidant enzymes and malondialdehyde content indicating oxidative damage and lipid peroxidation in the algal cells. Our findings reveal that triazophos has potential toxicity and environmental risks to one of the primary producers green algae.

Long noncoding RNA LINC00052 inhibits colorectal cancer metastasis by sponging microRNA-574-5p to modulate CALCOCO1 expression.

The dysregulation of long-chain noncoding ribonucleic acid (lncRNA) is a common phenomenon in many human cancers. Some studies on the biological function of long intergenic non-protein-coding RNA 52 (LINC00052) in cancer indicate that this gene can act as either oncogene or tumor suppressor in some kinds of cancers, such as breast cancer, gastric cancer, liver cancer, and lung cancer. However, the biological function of LINC00052 in colorectal cancer (CRC) has not been studied. Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) and Western blot (WB) techniques were applied to detect the expression levels of LINC00052, miR-574-5p, and calcium-binding and coiled-coil domain 1 (CALCOCO1) in CRC cells and tissues. We authenticated the biological function of LINC00052 and miR-574-5p in CRC, and find some target genes for LINC00052 and miR-574-5p via bioinformatics methods. Dual-luciferase reporter gene assay was performed to identify the interaction between LINC00052 and miR-574-5p or CALCOCO1 and miR-574-5p. The results demonstrated that LINC00052 was downregulated in CRC tissues compared with their adjacent tissues. And LINC00052 could suppress CRC cells metastasis both in vivo and in vitro. Beyond that, miR-574-5p was upregulated in CRC tissues, and as an oncogene, it accelerated CRC cell migration and invasion. More importantly, the results of our research demonstrated that LINC00052 could regulate the expression of CALCOCO1 via sponging miR-574-5p in CRC. Overall, our study illuminated the lncRNA-miRNA functional networks in CRC, and these results might provide a new research direction for the diagnosis and treatment of CRC.

A novel ferritin gene from Procambarus clarkii involved in the immune defense against Aeromonas hydrophila infection and inhibits WSSV replication.

Ferritin is a protein related to the storage of iron and widely distributed in animals. It participates in many biological process, including antioxidation, cell activation, angiogenesis, regulation of iron metabolic balance and immune defense. In the present study, a novel ferritin gene was identified from red swamp crayfish Procambarus clarkii, with a cDNA sequence encoding a predicted 221 amino-acid residues. The ferritin protein contains a 19-residue signal peptide and 145-residue classic ferritin domain. The NJ phylogenetic analysis showed PcFer clustered with other crustacean peptides. The recombinant PcFer protein was produced and purified in E. coli, and the anti-rabbit polyclonal antibody was obtained. The rPcFer exhibited iron binding activity at a dose-dependent effect. The qPCR and western blot analysis revealed that PcFer was highly expressed in hemocytes, hepatopancreas, and gills. After challenged with WSSV and Aeromonas hydrophila, the mRNA and protein expression patterns of PcFer were significantly up-regulated in hemocytes and hepatopancreas. dsRNA interfering technique was utilized to silence the expression of PcFer gene. The WSSV copy number in PcFer silenced shrimp was much higher than that in the control group. The present study indicated that PcFer was involved in the immune defense against WSSV and Aeromonas hydrophila, and might inhibit WSSV replication in P. clarkii. These results will provide important data support for further study of the functional role of the ferritin gene.

Effect of acute exposure of triazophos on histological structure and apoptosis of the brain and liver of zebrafish (Danio rerio).

Triazophos (TAP) has become a part of widespread pollutant of the aquatic environment due to its residue. Current study was designed to investigate the toxic effect of TAP at different doses (0.06, 0.3 and 1.5 mg/L) to the model organism of zebrafish (Danio rerio) by using multi-endpoint analysis in a 96 h acute exposure test. The direct observation that histological and ultrastructural alteration of zebrafish brain and liver were carried out via paraffin section in hematoxylin and eosin (H&E) staining and transmission electron microscopy (TEM), respectively. In addition, a series of methods were applied for exploring the physiological parameters related to cellular apoptosis. Results indicated that vacuolar structure after 96 h treatment with TAP were appeared in the molecular and granular layers of cerebellum. A large number of nuclear retraction, tissues vacuolation and cytoplasmic loss were observed in liver at histological level. From the fine structural level, the mitochondrial vacuolation and membrane damage of brain cells were found and the cristae of mitochondria disintegrated partly in hepatocytes. Onset of such histological structure alterations were one of the most intuitive reflection to TAP exposure, which needs to analyze biochemical alterations for further study. The mitochondrial membrane potential (MMP) showed a downward trend in the brain and liver of zebrafish. Simultaneously, the activity of caspase-3 and caspase-9 increased after 96 h exposure with a concentration-dependent manner, which could be served as a suitable indicator of cellular apoptosis. Furthermore, apoptosis-related genes (Apaf-1, p53, Bax, Bcl-2, caspase-3 and caspase-9) transcription showed different alterations in response to the TAP treatment. These results indicated that TAP exposure led to apoptosis in zebrafish brain and liver and it was speculated that the apoptosis may occur through mitochondrial pathway. The present study demonstrated that the exposure of zebrafish to the insecticide TAP led to observe its effects at both histological structure and apoptosis level in liver and brain.

Acute toxicity of triflumizole to freshwater green algae Chlorella vulgaris.

Triflumizole is one of imidazole fungicides that works by inhibiting ergosterol biosynthesis, and is widely used for the control of powdery mildew and scabs on various fruits and crops. Triflumizole residue has been frequently detected in soil and aquatic ecosystems. While many studies have focused on its toxic effect on terrestrial and aquatic animals, little attention has been paid to aquatic algae, the primary producers of aquatic environments. Therefore, we evaluated the acute (96 h) toxicity effects of triflumizole on the freshwater algae Chlorella vulgaris, by examining growth, cell morphology, photosynthesis, and oxidative stress. The results showed that the 96 h median inhibition concentration (96 h-EC50) was 0.82 mg/L (95% confidential interval 0.70-0.98 mg/L).The growth of algal cells was conspicuously inhibited by triflumizole exposure, and the cell surfaces appeared to be shrunkThe chlorophyll content (including Chl-a, Chl-b and T-Chl) dramatically decreased at triflumizole concentrations of 0.2 and 1.0 mg/L. In addition, the transcript abundance of photosynthesis-related genes (psaB, psbC and rbcL) showed obvious decreases in above treatments after 96 h of exposure to triflumizole. Moreover, the algal growth inhibition was accompanied by an increase in intracellular reactive oxygen species and malondialdehyde content, as well as increased activity of antioxidant enzymes such as superoxide dismutase and peroxidase, indicating oxidative stress and lipid peroxidation. Our findings reveal that triflumizole has potential toxicity to the primary producers (freshwater algae) in aquatic ecosystems.

Down-regulating ribonuclease inhibitor enhances metastasis of bladder cancer cells through regulating epithelial-mesenchymal transition and ILK signaling pathway.

Accumulating evidences implicate that ribonuclease inhibitor (RI) plays a suppressing role in cancer development. However, the mechanisms underlying antitumor of RI remain largely unknown. Epithelial-mesenchymal transition (EMT) is regarded as a key event in tumor progression. The reports have demonstrated that EMT was implicated in metastasis of bladder cancer. Therefore, we suppose that RI might involve regulating EMT of bladder cancer. Here bladder cancer T24 cells were transfected with pGensil-1-siRNA-RI vectors. HE staining, living cell observation, Phalloidine-FITC staining of microfilament, cell adhesion, scratch migration, and Matrigel invasion were examined respectively. RI expression and colocalization with ILK were detected using confocal microscope. Proteins associated with EMT were determined with Western blotting and immunohistochemistry in vivo and in vitro. Effects of RI expression on tumor growth, metastasis and EMT related proteins in BALB/C nude mouse and clinical human bladder cancer specimens were valued with histological, immunohistochemical and immunofluorescent examination respectively. We demonstrated that down-regulating RI increased cell proliferation, migration and invasion, changed cell morphology and adhesion, and rearranged cytoskeleton by inducing EMT and ILK signaling pathway in bladder cancer cells. In addition, we showed that down-regulating RI promoted tumorigenesis and metastasis of bladder cancer in vivo. Finally, we found that bladder cancer with invasive capability had higher Vimentin, Snail, Slug and Twist as well as lower E-cadherin and RI expression in clinical human specimens. Our results suggest that RI could play a novel role in inhibiting metastasis of bladder through regulating EMT and ILK signaling pathway.

Simultaneous determination of thirteen aminoalcohol-diterpenoid alkaloids in the lateral roots of Aconitum carmichaeli by solid-phase extraction-liquid chromatography-tandem mass spectrometry.

Aminoalcohol-diterpenoid alkaloids have been reported as the cardioactive components in the lateral roots of Aconitum carmichaeli (Fuzi) according to recent studies. Determination of these effective components is of great significance for quality control purposes for Fuzi. Here we report, for the first, the development and validation of a new method to determine the 13 aminoalcohol-diterpenoid alkaloids in Fuzi by using a simple and accurate solid-phase extraction-liquid chromatography-tandem mass spectrometry. The chromatographic analysis was performed on an ODS column with methanol-0.1 % formic acid (80 : 20, v/v) as the mobile phase. The quantification was performed using MS/MS detection in the positive ion mode with multiple reaction monitoring. Linearity was observed within a range of concentrations of 20-2,000 ng/mL. For all the analytes, the r value was greater than 0.9990. The limit of detection and the limit of quantitation were less than 0.5 ng/mL and 2.0 ng/mL, respectively. The intraday and interday precisions were less than 5% and 10%, respectively. The accuracy was within the range of 90 to 105%. This method was successfully applied to determine the 13 aminoalcohol-diterpenoid alkaloids in Fuzi from different origins and with different processing methods.

A novel role of ribonuclease inhibitor in regulation of epithelial-to-mesenchymal transition and ILK signaling pathway in bladder cancer cells.

Human ribonuclease inhibitor (RI) is a cytoplasmic acidic protein possibly involved in biological functions other than the inhibition of RNase A and angiogenin activities. We have previously shown that RI can inhibit growth and metastasis in some cancer cells. Epithelial-mesenchymal transition (EMT) is regarded as the beginning of invasion and metastasis and has been implicated in the metastasis of bladder cancer. We therefore postulate that RI regulates EMT of bladder cancer cells. We find that the over-expression of RI induces the up-regulation of E-cadherin, accompanied with the decreased expression of proteins associated with EMT, such as N-cadherin, Snail, Slug, vimentin and Twist and of matrix metalloprotein-2 (MMP-2), MMP-9 and Cyclin-D1, both in vitro and in vivo. The up-regulation of RI inhibits cell proliferation, migration and invasion, alters cell morphology and adhesion and leads to the rearrangement of the cytoskeleton in vitro. We also demonstrate that the up-regulation of RI can decrease the expression of integrin-linked kinase (ILK), a central component of signaling cascades controlling an array of biological processes. The over-expression of RI reduces the phosphorylation of the ILK downstream signaling targets p-Akt and p-GSK3ß in T24 cells. We further find that bladder cancer with a high-metastasis capability shows higher vimentin, Snail, Slug and Twist and lower E-cadherin and RI expression in human clinical specimens. Finally, we provide evidence that the up-regulation of RI inhibits tumorigenesis and metastasis of bladder cancer in vivo. Thus, RI might play a novel role in the development of bladder cancer through regulating EMT and the ILK signaling pathway.

Role of the long non-coding RNA LINC00052 in tumors.

Long intergenic non-protein coding RNA 52 (LINC00052) is a non-coding RNA with >200 nucleotides in length, which exerts important effects on several physiological and pathological processes of the human body. Recent studies have demonstrated that LINC00052 plays key roles in the tumorigenesis, progression and metastasis of multiple types of human cancer, including hepatocellular carcinoma, breast cancer, colorectal cancer, cervical carcinoma and gastric cancer. However, the associations between LINC00052 and these tumors remain unclear. The present review summarizes the biological functions of LINC00052 during the pathogenic process of certain tumors, and discusses its potential therapeutic targets.

An "on-off" electrochemiluminescence immunosensor for PIVKA-II detection based on the dual quenching of CeO2-Au-g-C3N4 hybrids by Ag nanocubes-VB2.

Herein, we report a novel dual-quenching electrochemiluminescence (ECL) immunosensor for detecting protein induced by vitamin K absence or antagonist-II (PIVKA-II) based on ECL resonance energy transfer (ECL-RET). In this protocol, self-accelerated ECL hybrids of CeO2 and Au nanoparticles functionalized g-C3N4 nanosheets (CeO2-Au-g-C3N4) were prepared, which exhibited high ECL emission in the presence of S2O82- as a coreactant for "signal on" state. Concretely, CeO2 with a reproducible redox couple of Ce3+ and Ce4+ could act as an efficient co-reaction accelerator to generate more oxidizing intermediate (SO4•-) to significantly self-promote the ECL emission of g-C3N4 NSs/S2O82- ECL system. Besides, Au nanoparticles not only accelerated electron transfer in the ECL process, but also provided massive active sites for biomolecules immobilization. The dual quenching labels of Ag nanocubes modified with vitamin B2 (AgNCs-VB2) were firstly proposed towards g-C3N4 NSs/S2O82- ECL system by ECL-RET, resulting in the remarkable ECL decrease for "signal off" state. Based on the sandwich immunoreaction, the "on-off" PIVKA-II ECL immunosensor gratifyingly possessed excellent detection sensitivity with the linear range of 0.4 pg mL-1-10 ng mL-1 and the low detection limit of 28.46 fg mL-1 (S/N = 3). This presented strategy might provide a potential alternative tool for PIVKA-II detection in medical research and early clinical diagnostics of hepatocellular carcinoma.

Water soluble multi-walled carbon nanotubes enhance peritoneal macrophage activity in vivo.

Due to their unique physical properties, carbon nanotubes are becoming promising novel materials in diverse areas like information technology, ultrastiff materials, biomedicine etc. The toxicological study of these materials is very imperative for the safety assessment in respect to their wide applications. The objective of the toxicity study was to find out whether water soluble multi-walled carbon nanotubes (S-MWNTs) would impair the phagocytic activity of macrophages, which play an important role in defenses against invading microbioles. The results showed that S-MWNTs did not impair the phagocytosis of macrophages; to our surprise, S-MWNT significantly enhanced this function. Increased activity of enzymes in lysosome also showed that S-MWNTs enhanced the lysosome function of macrophages. However, S-MWNTs can not influence nitric oxide secretion in macrophages and induce inflammation.

Haematological and blood biochemical characteristics of Glyptosternum maculatum (Siluriformes: Sisoridae) in Xizang (Tibet).

This study investigated the haematological and blood biochemical characteristics of Glyptosternum maculatum. The haematological and biochemical parameters were measured in 30 adult fish collected from Nyingchi Reach of Yarlung Zangbo River in Tibet. The red blood cell count (RBC), haemoglobin concentration (Hb), haematocrit (Hct), erythrocyte osmotic fragility (maxEof and minEof), the erythrocyte sedimentation rate, mean cell volume (MCV), mean cellular haemoglobin content (MCH), and mean cell haemoglobin concentration (MCHC) were determined. Compared with other Siluriformes fishes, G. maculatum showed similar mean values for Hct, Hb, MCH, and MCHC and had slightly lower RBC and higher MCV. The biochemical parameters were assayed including alanine aminotransferase, aspartate aminotransferase (AST), alkaline phosphatase, total protein, albumin, globulin, albumin/globulin ratio, total bilirubin, direct bilirubin, urea, creatinine, glucose, total cholesterol, and triglyceride. The result showed that the value of AST in G. maculatum was obviously higher than that in Rhamdia quelen as well as in Silurus merdionalis.

[Spectroscopic investigation on the interaction between astragalin from lotus leaf and DNA].

The interaction between astragalin (AST) from lotus leaf and deoxyribonucleic acid (DNA) in Tris-HCl buffer (pH = 7.4) was investigated by the application of fluorescence spectroscopy and ultraviolet absorption spectroscopy, the effects of ionic strength and anion quencher KI on the fluorescence intensity of AST from lotus leaf and the system of AST-DNA were explored, and the competitive binding to DNA between AST from lotus leaf and Neutral Red(NR)dye was also studied. The results demonstrated that AST could bind to DNA and the formed complex quenched the intrinsic fluorescence of AST from lotus leaf through static quenching mechanism. The quenching rate constants of biomolecule(Kq)of the reaction of DNA with AST from lotus leaf were calculated to be 3.120 X 10(12) and 2.630 X 10(12) L x mol(-1) x s(-1) by Stern-Volumer equation, the corresponding binding constants (Kd) were computed to be 3.412 x 10(12) and 1.762 x 10(4) L x mol(-1) and the number of binding sites(n) was counted to be 1.007 and 0.962 between AST from lotus leaf and DNA at 298 and 308 K, respectively. When bound to DNA, the AST from lotus leaf showed hypochromic effect and red shift in the absorption spectra. It was also found that different ionic strength had little or no effect on the fluorescence intensity of AST and AST-DNA, but the fluorescence intensity of AST-DNA quenched by anionic quencher KI was much less than that of free AST. AST could be intercalated into DNA and displaced the NR from the NR-DNA complex. It was showed that AST from lotus leaf could combine with DNA in the mode of intercalation.

Induction of time- and dose-dependent oxidative stress of triazophos to brain and liver in zebrafish (Danio rerio).

Extensive use of triazophos for the chemical control of pests in agriculture or aquaculture might strongly disturb the aquatic environment due to residue accumulation through various routes like surface run-off, spray-drift and effluent from factories, which have potential negative effects to non-target aquatic organisms. Previous studies have documented the antioxidative effects of triazophos to mammals, however, the oxidative toxicity of triazophos to fish has not been adequately studied to date. Thus, an acute exposure (96 h) to triazophos at different concentrations of 0.06, 0.3 and 1.5 mg/L (corresponding to 1/50th, 1/10th and 1/2th of 96 h-LC50, respectively), was conducted to investigate the triazophos-induced oxidative stress in adult zebrafish (Danio rerio). The results showed that the time- and dose-dependent induction of oxidative stress except for the ROS level in liver after 24 and 48 h exposure, as indicated by increased reactive oxygen species (ROS), malondialdehyde (MDA) level and a compromised antioxidant defense system, including increased superoxide dismutase (SOD) activity, catalase (CAT) activity, glutathione (GSH) content as well as the increased at first and decreased afterwards genes expression (Sod1, Sod2, Cat and Gpx) in brain. Simultaneously, ROS and MDA showed an increased trend, SOD activity, CAT activity and GSH content showed a trend of increasing at 24 h and decreasing at 48 h, and then increasing at 96 h and Sod1, Sod2, Cat and Gpx gene showed decreasing at first and then increasing in liver tissue. The present study concluded that the damage of the antioxidant system by triazophos induced oxidative stress in the brain and liver of zebrafish with concomitant lipid peroxidation, which is an important mechanism underlying the triazophos-induced acute toxicity.