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Environmental Toxicology and... Jun 2016This study was to investigate the combined toxicity of silica nanoparticles (SiNPs) and methylmercury (MeHg) on cardiovascular system in zebrafish (Danio rerio) embryos....
This study was to investigate the combined toxicity of silica nanoparticles (SiNPs) and methylmercury (MeHg) on cardiovascular system in zebrafish (Danio rerio) embryos. Ultraviolet absorption analysis showed that the co-exposure system had high absorption and stability. The dosages used in this study were based on the NOAEL level. Zebrafish embryos exposed to the co-exposure of SiNPs and MeHg did not show any cardiovascular malformation or atrioventricular block, but had an inhibition effect on bradycardia. Using o-Dianisidine for erythrocyte staining, the cardiac output of zebrafish embryos was decreased gradually in SiNPs, MeHg, co-exposure groups, respectively. Co-exposure of SiNPs and MeHg enhanced the vascular endothelial damage in Tg(fli-1:EGFP) transgenic zebrafish line. Moreover, the co-exposure significantly activated the oxidative stress and inflammatory response in neutrophils-specific Tg(mpo:GFP) transgenic zebrafish line. This study suggested that the combined toxic effects of SiNPs and MeHg on cardiovascular system had more severe toxicity than the single exposure alone.
Topics: Animals; Animals, Genetically Modified; Cardiovascular System; Drug Synergism; Embryo, Nonmammalian; Erythrocytes; Methylmercury Compounds; Nanoparticles; No-Observed-Adverse-Effect Level; Reactive Oxygen Species; Silicon Dioxide; Zebrafish
PubMed: 27163730
DOI: 10.1016/j.etap.2016.05.004 -
Aquatic Toxicology (Amsterdam,... Jun 2016Copper, as an essential trace mineral, can cause diseases such as childhood leukemia at excess levels, but has been applied in anemia therapy for a long time. However,...
Copper, as an essential trace mineral, can cause diseases such as childhood leukemia at excess levels, but has been applied in anemia therapy for a long time. However, few reports have studied its role during hematopoiesis at the molecular level in an animal model. In this study, by microarray, qRT-PCR, whole-mount in situ hybridization and O-dianisidine staining detections, we revealed the increased expression of hemoglobin in copper-exposed embryos. Secondly, we found that copper-exposed embryos exhibited high levels of reactive oxygen species (ROS), and genes in oxygen binding and oxygen transporting were up-regulated in the embryos. Finally, we found that ROS scavengers NAC, GSH, and DMTU not only inhibited in vivo ROS levels induced by copper, but also significantly decreased high expression of hemoglobin back to almost normal levels in copper exposed embryos, and also helped with copper elimination from the embryos. Our data first demonstrated that ROS mediated copper induced hemoglobin expression in vertebrates, partly revealing the underlying molecular mechanism of copper therapy for anemia. Moreover, we revealed that copper homeostasis was broken by its induced ROS and ROS helped with copper overloading in the body, which could be applied as a novel therapy target for copper-caused diseases.
Topics: Analysis of Variance; Animals; Copper; Gene Expression Regulation; Hemoglobins; Mass Spectrometry; Microarray Analysis; Polymerase Chain Reaction; Reactive Oxygen Species; Reverse Transcriptase Polymerase Chain Reaction; Up-Regulation; Water Pollutants, Chemical; Zebrafish
PubMed: 26991749
DOI: 10.1016/j.aquatox.2016.03.008 -
Naunyn-Schmiedeberg's Archives of... May 2016Melatonin is known as a strong antioxidant and possesses anti-inflammatory properties. Recently, melatonin was shown to improve colitis in animal models of inflammatory...
Melatonin is known as a strong antioxidant and possesses anti-inflammatory properties. Recently, melatonin was shown to improve colitis in animal models of inflammatory bowel diseases. The aim of the present study was to characterize the role of melatonin receptors (MT) in the anti-inflammatory effect of melatonin and to assess the anti-inflammatory potential of two novel MT receptor agonists, Neu-P11 and Neu-P67, in the mouse model of trinitrobenzenesulfonic acid (TNBS)-induced colitis. Colitis was induced on day 1 by intracolonic (i.c.) administration of TNBS in 30 % ethanol in saline. Melatonin (4 mg/kg, per os (p.o.)), Neu-P11 (20 mg/kg, p.o.; 50 mg/kg, intraperitoneally (i.p.), 50 mg/kg, i.c.), and Neu-P67 (20 mg/kg, p.o.) were given twice daily for 3 days. Luzindole (5 mg/kg, i.p.) was injected 15 min prior to melatonin administration. On day 4, macroscopic and microscopic damage scores were assessed and myeloperoxidase (MPO) activity quantified using O-dianisidine-based assay. Melatonin significantly attenuated colitis in mice, as indicated by the macroscopic score (1.90 ± 0.34 vs. 3.82 ± 0.62 for melatonin- and TNBS-treated mice, respectively), ulcer score (0.87 ± 0.18 vs. 1.31 ± 0.19, respectively), and MPO activity (4.68 ± 0.70 vs.6.26 ± 0.94, respectively). Luzindole, a MT receptor antagonist, did not inhibit the anti-inflammatory effect of melatonin (macroscopic score 1.12 ± 0.22, ulcer score 0.50 ± 0.16); however, luzindole increased MPO activity (7.57 ± 1.05). MT receptor agonists Neu-P11 and Neu-P67 did not improve inflammation induced by TNBS. Melatonin, but not MT receptor agonists, exerts potent anti-inflammatory action in acute TNBS-induced colitis. Our data suggests that melatonin attenuates colitis by additional, MT receptor-independent pathways.
Topics: Animals; Anti-Inflammatory Agents; Colitis; Colon; Indoles; Male; Melatonin; Mice, Inbred BALB C; Peroxidase; Pyrans; Receptors, Melatonin; Trinitrobenzenesulfonic Acid
PubMed: 26899972
DOI: 10.1007/s00210-016-1214-x -
Zhonghua Yi Xue Za Zhi Jan 2016To investigate the effects of fine particulate matter with a mean aerodynamic diameter ≤2.5 μm (PM2.5) collected from Lanzhou city on phagocytic function of alveolar...
OBJECTIVE
To investigate the effects of fine particulate matter with a mean aerodynamic diameter ≤2.5 μm (PM2.5) collected from Lanzhou city on phagocytic function of alveolar macrophages (AM) in chronic obstructive pulmonary disease (COPD) mice.
METHODS
Forty male mice were randomly divided into four groups: healthy group, healthy PM2.5 group, COPD group and COPD PM2.5 group. COPD mice were established by cigarette smoking. PM2.5 (10 mg/kg) collected by air sampler was intratracheally instilled in healthy PM2.5 group and COPD PM2.5 group. Mice were sacrificed after 14 days, and alveolar macrophages (AM) were isolated. Mean fluorescence intensity (MFI) and the positive percent of alveolar macrophages engulfing flurescein isothiocyanate-labeled Escherichia coli (FITC-E.coli) (AM%) were detected by flow cytometry. Total antioxidative capacity (TAC) was measured by O-phenanthroline colorimetry. Malondialdehyde (MDA) was measured by thiobarbiturieacid colorimetry and myeloperoxidase (MPO) was measured by O-dianisidine colorimetry.
RESULTS
The peak inspiratory flow (PIF), peak expiratory flow (PEF) and dynamic compliance (Cdyn) of COPD group were significantly lower than healthy control group. The pathology of COPD group showed disruption of alveolar septa, formation of emphysema, and that the number of alveoli had a significant reduction. The MFI and AM% in COPD group were significant lower than healthy group (14.1±1.7 vs 43.2±6.1, 9.2%±2.3% vs 69.1%±8.3%)(all P<0.01). Comparing to healthy group and COPD group, the MFI and AM% in healthy PM2.5 group (20.3±4.5, 40.4%±4.4%) and COPD PM2.5 group (7.5±1.3, 6.0%±2.2%) were respectively lowered. The level of TAC in COPD group was significantly lower than healthy group [(3.10±0.64) vs (15.43±0.69)U/mg], the levels of MDA and MPO in COPD group were higher than healthy group[(2.72±0.13) vs (1.31±0.16) nmol/mg, (1.63±0.11) vs (0.92±0.13)U/g] (all P<0.01). In both healthy PM2.5 group and COPD PM2.5 group, the levels of TAC [(6.75±1.06), (2.34±0.61) U/mg] were lower than their corresponding control group; while the levels of MDA [(1.96±0.31), (3.20±0.19) nmol/mg] and the levels of MPO [(1.01±0.19), (1.74±0.13) U/g] were increased (all P<0.01). For the COPD group at baseline and after the intervention of PM2.5, the MFI and AM% showed positive correlation with the levels of TAC, and negative correlation with the levels of MDA , and negative correlation with the levels of MPO (all P<0.05). For health group at baseline and after the intervention PM2.5, the above relationships still existed (all P<0.05).
CONCLUSION
PM2.5 can damage phagocytosis of AM and exacerbate oxidative stress in COPD mice, and AM phagocytosis impairment by PM2.5 is closely associated with oxidative stress.
Topics: Animals; Lung; Macrophages, Alveolar; Male; Malondialdehyde; Mice; Oxidative Stress; Particulate Matter; Phagocytosis; Pulmonary Alveoli; Pulmonary Disease, Chronic Obstructive; Pulmonary Emphysema; Smoking; Nicotiana
PubMed: 26879794
DOI: 10.3760/cma.j.issn.0376-2491.2016.04.016 -
Iranian Journal of Microbiology Feb 2015Due to the evolution of multidrug-resistant strains, screening of natural resources, especially actinomycetes, for new therapeutic agents discovery has become the...
BACKGROUND AND OBJECTIVE
Due to the evolution of multidrug-resistant strains, screening of natural resources, especially actinomycetes, for new therapeutic agents discovery has become the interests of researchers. In this study, molecular, chemical and biological screening of soil actinomycetes was carried out in order to search for peptide-producing actinomycetes.
MATERIALS AND METHODS
60 actinomycetes were isolated from soils of Iran. The isolates were subjected to molecular screening for detection NRPS (non-ribosomal peptide synthetases) gene. Phylogenic identification of NRPS containing isolates was performed. Chemical screening of the crude extracts was performed using chlorine o-dianisidine as peptide detector reagent and bioactivity of peptide producing strains was determined by antimicrobial bioassay. High pressure liquid chromatography- mass spectrometry (HPLC-MS) with UV-visible spectroscopy was performed for detection of the metabolite diversity in selected strain.
RESULTS
Amplified NRPS adenylation gene (700 bp) was detected among 30 strains. Phylogenic identification of these isolates showed presence of rare actinomycetes genera among the isolates and 10 out of 30 strains were subjected to chemical screening. Nocardia sp. UTMC 751 showed antimicrobial activity against bacterial and fungal test pathogens. HPLC-MS and UV-visible spectroscopy results from the crude extract showed that this strain has probably the ability to produce new metabolites.
CONCLUSION
By application of a combined approach, including molecular, chemical and bioactivity analysis, a promising strain of Nocardia sp. UTMC 751 was obtained. This strain had significant activity against Staphylococcus aureus and Pseudomonas aeruginosa. Strain Nocardia sp. UTMC 751 produce five unknown and most probably new metabolites with molecular weights of 274.2, 390.3, 415.3, 598.4 and 772.5. This strain had showed 99% similarity to Nocardia ignorata DSM 44496 T.
PubMed: 26644870
DOI: No ID Found -
Nanotoxicology 2016The toxicity mechanism of nanoparticles on vertebrate cardiovascular system is still unclear, especially on the low-level exposure. This study was to explore the toxic...
The toxicity mechanism of nanoparticles on vertebrate cardiovascular system is still unclear, especially on the low-level exposure. This study was to explore the toxic effect and mechanisms of low-dose exposure of silica nanoparticles (SiNPs) on cardiac function in zebrafish embryos via the intravenous microinjection. The dosage of SiNPs was based on the no observed adverse effect level (NOAEL) of malformation assessment in zebrafish embryos. The mainly cardiac toxicity phenotypes induced by SiNPs were pericardial edema and bradycardia but had no effect on atrioventricular block. Using o-Dianisidine for erythrocyte staining, the cardiac output of zebrafish embryos was decreased in a dose-dependent manner. Microarray analysis and bioinformatics analysis were performed to screen the differential expression genes and possible pathway involved in cardiac function. SiNPs induced whole-embryo oxidative stress and neutrophil-mediated cardiac inflammation in Tg(mpo:GFP) zebrafish. Inflammatory cells were observed in atrium of SiNPs-treated zebrafish heart by histopathological examination. In addition, the expression of TNNT2 protein, a cardiac contraction marker in heart tissue had been down-regulated compared to control group using immunohistochemistry. Confirmed by qRT-PCR and western blot assays, results showed that SiNPs inhibited the calcium signaling pathway and cardiac muscle contraction via the down-regulated of related genes, such as ATPase-related genes (atp2a1l, atp1b2b, atp1a3b), calcium channel-related genes (cacna1ab, cacna1da) and the regulatory gene tnnc1a for cardiac troponin C. Moreover, the protein level of TNNT2 was decreased in a dose-dependent manner. For the first time, our results demonstrated that SiNPs induced cardiac dysfunction via the neutrophil-mediated cardiac inflammation and cardiac contraction in zebrafish embryos.
Topics: Animals; Dose-Response Relationship, Drug; Embryo, Nonmammalian; Heart; Heart Function Tests; Inflammation; Myocardial Contraction; Nanoparticles; Neutrophils; Oxidative Stress; Silicon Dioxide; Zebrafish
PubMed: 26551753
DOI: 10.3109/17435390.2015.1102981 -
International Journal of Biological... Nov 2015Suicide inactivation is a common mechanism observed for haem peroxidases, in which the enzyme is inactivated as a result of self-oxidation mediated by intermediate...
Suicide inactivation is a common mechanism observed for haem peroxidases, in which the enzyme is inactivated as a result of self-oxidation mediated by intermediate highly oxidizing enzyme forms during the catalytic cycle. The time-dependence and the inactivation mechanism of Cytisus multiflorus peroxidase (CMP) by hydrogen peroxide were studied kinetically with four co-substrates (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), ferulic acid, guaiacol and o-dianisidine). Catalytic activity decreased following the sequence ABTS>guaiacol>ferulic acid>o-dianisidine. Once the intermediate complex (compound III-H2O2) had been formed, competition was established between the catalytic pathway and the suicide inactivation pathway. One mole of CMP afforded around 3790 turnovers of H2O2 for ABTS before its complete inactivation. These results suggest that CMP follows a suicide mechanism, the enzyme not being protected in this case. The mechanism of suicide inactivation is discussed with a view to establishing a broad knowledge base for future rational protein engineering.
Topics: Cytisus; Enzyme Activation; Hydrogen Peroxide; Kinetics; Least-Squares Analysis; Time Factors
PubMed: 26407901
DOI: 10.1016/j.ijbiomac.2015.09.033 -
Pharmacological Research Dec 2015The zebrafish (Danio rerio) is a very popular vertebrate model system, especially embryos represent a valuable tool for in vivo pharmacological assays. This is mainly...
The zebrafish (Danio rerio) is a very popular vertebrate model system, especially embryos represent a valuable tool for in vivo pharmacological assays. This is mainly due to the zebrafish advantages when compared to other animal models. Erythropoietin is a glycoprotein hormone that acts principally on erythroid progenitors, stimulating their survival, proliferation and differentiation. Recombinant human erythropoietin (rhEPO) has been widely used in medicine to treat anemia and it is one of the best-selling biotherapeutics worldwide. The recombinant molecule, industrially produced in CHO cells, has the same amino acid sequence of endogenous human erythropoietin, but differs in the glycosylation pattern. This may influence efficacy and safety, particularly immunogenicity, of the final product. We employed the zebrafish embryo as a vertebrate animal model to perform in vivo pharmacological assays. We conducted a functional analysis of rhEPO alpha Eprex(®) and two biosimilars, the erythropoietin alpha Binocrit(®) and zeta Retacrit(®). By in silico analysis and 3D modeling we proved the interaction between recombinant human erythropoietin and zebrafish endogenous erythropoietin receptor. Then we treated zebrafish embryos with the 3 rhEPOs and we investigated their effect on erythrocytes production with different assays. By real time-PCR we observed the relative upregulation of gata1 (2.4 ± 0.3 fold), embryonic α-Hb (1.9 ± 0.2 fold) and β-Hb (1.6 ± 0.1 fold) transcripts. A significant increase in Stat5 phosphorylation was also assessed in embryos treated with rhEPOs when compared with the negative controls. Live imaging in tg (kdrl:EGFP; gata1:ds-red) embryos, o-dianisidine positive area quantification and cyanomethemoglobin content quantification revealed a 1.8 ± 0.3 fold increase of erythrocytes amount in embryos treated with rhEPOs when compared with the negative controls. Finally, we verified that recombinant human erythropoietins did not cause any inflammatory response in the treated embryos. Our data showed that zebrafish embryo can be a valuable tool to study in vivo effects of complex pharmacological compounds, such as recombinant human glycoproteins, allowing to perform fast and reproducible pharmacological assays with excellent results.
Topics: Amino Acid Sequence; Animals; Biosimilar Pharmaceuticals; Computational Biology; Embryo, Nonmammalian; Epoetin Alfa; Erythropoietin; GATA1 Transcription Factor; Humans; Models, Animal; Molecular Sequence Data; Receptors, Erythropoietin; Recombinant Proteins; Sequence Alignment; Up-Regulation; Zebrafish
PubMed: 26361727
DOI: 10.1016/j.phrs.2015.09.004 -
Drug Metabolism and Personalized Therapy Sep 2015Hemoglobin is released to the serum after erythrocyte lyses. Haptoglobin is responsible for carrying hemoglobin into the serum. In hemolytic disease, the amount of...
BACKGROUND
Hemoglobin is released to the serum after erythrocyte lyses. Haptoglobin is responsible for carrying hemoglobin into the serum. In hemolytic disease, the amount of hemoglobin which is released to the serum is high; however, the amount of haptoglobin is not enough for binding all the released hemoglobins. Free hemoglobin has peroxidase activity (a pseudoenzyme) and has been indicated to be harmful for patients. This study is focused on the effect of cimetidine on peroxidase activity of hemoglobin.
METHODS
Erythrocytes were lysed to obtain hemoglobin. Peroxidase activity of hemoglobin was detected using o-dianisidine and H(2)O(2) as substrates.
RESULTS
Our results showed that the drug operated as an activator for the pseudoenzyme. Cimetidine bound to the pseudoperoxidase in an un-competitive manner and decreased the Km. Half maximal effective concentration (EC(50)) of cimetidine was determined to be about 12.5 mM. Alkaline pH increased the rate of reaction. Arrhenius plot showed that the activation energies of reactions in the absence and presence of drug were about 10.5 kJ/mol and 7.65 kJ/mol, respectively.
CONCLUSIONS
The results demonstrated that cimetidine activates the peroxidase activity of free hemoglobin. Hence, it is suggested that the prescription of cimetidine for the patients with hemolyses diseases may enhance the harmful effects of free hemoglobin in these patients.
Topics: Anti-Ulcer Agents; Biocatalysis; Cimetidine; Enzyme Activation; Hemoglobin A; Histamine H2 Antagonists; Humans; Hydrogen-Ion Concentration; Peroxidases; Temperature
PubMed: 26167985
DOI: 10.1515/dmpt-2014-0032 -
Chemical Science Jul 2015As an alternative to Darwinian evolution relying on catalytic promiscuity, a protein may acquire auxiliary function upon metal binding, thus providing it with a novel...
As an alternative to Darwinian evolution relying on catalytic promiscuity, a protein may acquire auxiliary function upon metal binding, thus providing it with a novel catalytic machinery. Here we show that addition of cupric ions to a 6-phosphogluconolactonase bearing a putative metal binding site leads to the emergence of peroxidase activity ( 7.8 × 10 s, 1.1 × 10 M). Both X-ray crystallographic and EPR data of the copper-loaded enzyme reveal a bis-histidine coordination site, located within a shallow binding pocket capable of accommodating the -dianisidine substrate.
PubMed: 29218172
DOI: 10.1039/c5sc01065a