Characterization of titanium dioxide nanoparticles imprinted for tyrosine by flow field-flow fractionation and spectrofluorimetric analysis

Films based on TiO₂ nanoparticles (NPs) have been successfully used as sensing elements in chemical sensors. TiO₂ colloidal suspensions can be obtained by spontaneous hydrolysis in acidic solutions of Ti(IV) compounds. The obtained TiO₂ NPs can be employed to build up nanostructured films. With the purpose of preparing TiO₂-based nanostructured, imprinted materials as sensing elements for piezoelectric sensors, we obtained TiO₂ NP dispersions by hydrolyzing potassium titanyl oxalate in the presence of a target analyte (tyrosine). Since morphological properties of the synthesized NPs are known to influence the nanostructured film characteristics, an analytical strategy to characterize such colloidal systems can combine a size-based separation method with spectroscopic analysis to correlate the particle size distribution (PSD) with the particle-target interaction properties able to determine the sensing efficiency.In this work, we present the characterization of colloidal tyrosine-TiO₂ NP systems by flow field-flow fractionation (FlFFF) with online, UV/Vis absorption detection and offline fluorescence analysis. FlFFF eliminates the possible contribution of free tyrosine to the absorption and fluorescence properties of the NPs. FlFFF also fractionates NPs on a size basis. Particle size distribution (PSD) profiles of the fractionated NPs are then obtained by conversion of the multi-wavelength UV/Vis fractograms. Size of the fractionated NPs is finally related to fluorescence properties of the collected NPs fractions. Good correlation between the fluorescence intensity, which is proportional to the tyrosine uptake, and the FlFFF-based, NP mass-size frequency distribution finally confirms the existence of tyrosine-TiO₂ NP interaction.     

Effects of H2O2 on the growth, secretion, and metabolism of hybridoma cells in culture

Summary The effect of low concentrations of hydrogen peroxide (H₂O₂) (5 × 10⁻⁷−9.5 × 10⁻⁷ M) on cell growth and antibody production was investigated with murine hybridoma cells (Mark 3 and anti-hPL) in culture. Cell growth, measured by flow cytometry with morphological parameters, was significantly stimulated by H₂O₂ (8 × 10⁻⁷ M) but H₂O₂ concentration of 7 × 10⁻⁶ M and above increased cell death. H₂O₂ stimulation of antibody production was nonsignificant. The metabolism of cells treated with 8 × 10⁻⁷ or 1 × 10⁻⁵ M H₂O₂ was similar to that of the control in terms of glucose and glutamine consumption, lactate and ammonia production, and amino acid concentrations in the medium. The concentrations of lactate dehydrogenase, a marker of cell death, in test and control cells were similar. However, concentrations of intracellular free radicals measured by flow cytometry with dihydrorhodamine 123 (DHR 123) and dichlorofluorescein diacetate (DCFH-DA) as fluorochromes were different. The reactive oxygen species content of cells in 8 × 10⁻⁷ M H₂O₂ was similar to that of the controls, but there was a sudden, marked production of superoxide anions (detected with DHR 123) and H₂O₂ or peroxides (detected with DCFH-DA) by cells incubated with 1 × 10⁻⁵ M H₂O₂ which increased with increasing H₂O₂ until cell death.     

Assessment of titanium dioxide nanoparticles toxicity via oral exposure in mice: effect of dose and particle size

Objective: The aim of the present work is to evaluate the toxicity of titanium dioxide nanoparticles (TiO₂NPs) according to their doses and particle sizes.

Materials and methods: The effect of five days oral administration of TiO₂NPs (21 and 80?nm) with different doses (50, 250 and 500?mg/kg body weight) was assessed in mice via measurement of oxidative stress markers; glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), malondialdehyde (MDA) and nitric oxide (NO), liver function indices; aspartate and alanine aminotransferases (AST and ALT), chromosomal aberrations and liver histopathological pattern.

Results: The results revealed drastic alterations in all the measured parameters and showed positive correlation with the gradual dose increment. In addition, the smaller particle size of TiO₂NPS (21?nm) had more adverse effect in all the selected biochemical parameters, genetic aberrations and histological investigations.

Conclusions: Toxicity of TiO₂NPs increases in a dose-dependent manner and vice versa with particles size. The evaluated biomarkers are good indicators for TiO₂NPs toxicity. More detailed studies are required before the recommendation of TiO₂NP_S as food additives.     

Protective effect of ascorbic acid and glutathione on AlCl3-inhibited growth of rice roots

The effect of AlCl₃ on the antioxidant system of rice roots and the role of applied antioxidants ascorbic acid (AsA) and glutathione (GSH) in AlCl₃-inhibited growth of rice roots were investigated. AlCl₃ treatment resulted in a rapid inhibition of root growth but had no effect on lipid peroxidation and antioxidative enzyme activities in rice roots. AlCl₃ treatment resulted in lower content of H₂O₂, AsA, and GSH than in controls. Exogenous AsA or GSH counteracted growth inhibition of rice roots induced by AlCl₃. AlCl₃ treatment increased syringaldazine peroxidase (SPOX) activities and lignin content in rice roots. Exogenous AsA or GSH prevented the decrease in H₂O₂ content and the increase in SPOX activities and lignin content in rice roots caused by AlCl₃. Results suggest that lignification induced by low AsA or GSH content may explain the mechanism of Al-inhibited growth of rice roots.     

Dependence of H2O2 Formation by Rat Heart Mitochondria on Substrate Availability and Donor Age

We have examined the substrate specificity and inhibitor sensitivity of H₂O₂ formation by rat heart mitochondria. Active H₂O₂ production requires both a high fractional reduction of Complex I (indexed by NADH/NAD⁺ + NADH ratio) and a high membrane potential, . These conditions are achieved with supraphysiological concentrations of succinate. With physiological concentrations of NAD-linked substrates, rates of H₂O₂ formation are much lower (less than 0.1% of respiratory chain electron flux) but may be stimulated by the Complex III inhibitor antimycin A, but not by myxothiazol. Addition of Mn²⁺ to give 10 nmol/mg of mitochondrial protein enhances H₂O₂ production with all substrate combinations, possibly by repleting mitochondrial superoxide dismutase with this cation. Contrary to previously published work, no increased activity of H₂O₂ production was found with heart mitochondria from senescent (24 month) rats, relative to young adults (6 month).     

Superoxide dismutase (SOD) as a potential inhibitory mediator of inflammation via neutrophil apoptosis

Superoxide dismutase (SOD) is supposed to be an effective agent for neutrophil-mediated inflammation in the area of critical medicine. We investigated the involvement of SOD in the regulation of neutrophil apoptosis. Exogenously added SOD effectively induced neutrophil apoptosis, and the fluorescence patterns determined using annexin-V and the 7-AAD were similar to those seen in Fas-mediated neutrophil apoptosis. Neutrophils are short-lived leukocytes that need to be removed safely by apoptosis. The clearance of apoptotic neutrophils from sites of inflammation is a crucial determinant of the resolution of inflammation. Catalase inhibited the neutrophil apoptosis and caspase-3 activation. Spontaneous apoptosis, hydrogen peroxide and anti-Fas antibody-induced apoptosis of neutrophils were accelerated in Down's syndrome patients, in whom the SOD gene is overexpressed. Hydrogen peroxide was thought to be a possible major mediator of ROS-induced neutrophil apoptosis in caspase-dependent manner. Neutrophil apoptosis represents a crucial step in the mechanism governing the resolution of inflammation and has been suggested as a possible target for the control of neutrophil-mediated tissue injury. SOD may be a potential inhibitory mediator of neutrophil-mediated inflammation.     

ECS1参与调节CO2诱导的拟南芥气孔关闭和H2O2积累

CO2浓度升高可以诱导植物叶片气孔关闭,提高植物对高浓度CO2的适应性.但植物如何感知CO2浓度变化并启动气孔关闭反应的分子机制至今仍不十分清楚.利用高通量、非侵入的远红外成像技术,建立了拟南芥(Arabidopsis thaliana)气孔对CO2浓度变化反应相关的突变体筛选技术,筛选出对环境CO2浓度敏感的拟南芥突变体ecs1.遗传学分析表明,ecs1 为单基因隐性突变体,突变基因ECS1编码一个跨膜钙离子转运蛋白.与野生型拟南芥相比,360 μL·L-1CO2可引起ecs1突变体叶片温度上升和气孔关闭,ecs1突变体对900 μL·L-1CO2长时间处理具有较强的适应性.进一步的实验表明,360 μL·L-1CO2即可诱导ecs1突变体叶片积累较高浓度的H2O2,而900 μL·L-1CO2才能够诱导野生型拟南芥叶片积累H2O2.因此,ECS1可能参与调节高浓度CO2诱导的拟南芥气孔关闭和H2O2产生,H2O2可能作为第二信号分子介导CO2诱导拟南芥气孔关闭的反应.     

Photo-irradiated titanium dioxide catalyzes site specific DNA damage via generation of hydrogen peroxide

Titanium dioxide (TiO₂) is a potential photosensitizer for photodynamic therapy. In this study, the mechanism of DNA damage catalyzed by photo-irradiated TiO₂ was examined using [₃₂P]-5'-end-labeled DNA fragments obtained from human genes. Photo-irradiated TiO₂ (anatase and rutile) caused DNA cleavage frequently at the guanine residue in the presence of Cu(II) after E. coli formamidopyrimidine-DNA glycosylase treatment, and the thymine residue was also cleaved after piperidine treatment. Catalase, SOD and bathocuproine, a chelator of Cu(I), inhibited the DNA damage, suggesting the involvement of hydrogen peroxide, superoxide and Cu(I). The photocatalytic generation of Cu(I) from Cu(II) was decreased by the addition of SOD. These findings suggest that the inhibitory effect of SOD on DNA damage is due to the inhibition of the reduction of Cu(II) by superoxide. We also measured the formation of 8-oxo-7,8-dihydro-2' -deoxyguanosine, an indicator of oxidative DNA damage, and showed that anatase is more active than rutile. On the other hand, high concentration of anatase caused DNA damage in the absence of Cu(II). Typical free hydroxyl radical scavengers, such as ethanol, mannnitol, sodium formate and DMSO, inhibited the copper-independent DNA photodamage by anatase. In conclusion, photo-irradiated TiO₂ particles catalyze the copper-mediated site-specific DNA damage via the formation of hydrogen peroxide rather than that of a free hydroxyl radical. This DNA-damaging mechanism may participate in the phototoxicity of TiO₂.     

Utilization of H2O2 as the Oxygen Source by Bacteria of the Genera Pseudomonas and Rhodococcus

The growth of bacteria of the genera Pseudomonas and Rhodococcus in the presence of hydrogen peroxide as the sole source of oxygen was studied. The toxic effect of H₂O₂ in the concentration range of 100–200 g/ml was shown to extend the lag phase by two to three days. Apart from the peroxide toxicity, the bacterial growth was inhibited by the toxic effect of dissolved oxygen in concentrations over 100 g O₂/ml; in the presence of a liquid hydrocarbon phase, this effect was alleviated. Under decreased partial pressure of oxygen in the presence of hydrocarbons (12–15 vol %), culture growth was initiated at high initial concentrations of H₂O₂ (300 g/ml). When hydrogen peroxide concentrations exceeded 320 g/ml, no growth occurred, regardless of how much hydrocarbon was added.     

Antimicrobial photodynamic activity of toluidine blue encapsulated in mesoporous silica nanoparticles against Pseudomonas aeruginosa and Staphylococcus aureus

In the present study, the antimicrobial and antibiofilm efficacy of toluidine blue (TB) encapsulated in mesoporous silica nanoparticles (MSN) was investigated against Pseudomonas aeruginosa and Staphylococcus aureus treated with antimicrobial photodynamic therapy (aPDT) using a red diode laser 670?nm wavelength, 97.65?J cm^?2 radiant exposure, 5?min). Physico-chemical techniques (UV-visible (UV-vis) absorption, photoluminescence emission, excitation, and FTIR) and high-resolution transmission electron microscopy (HR-TEM) were employed to characterize the conjugate of TB encapsulated in MSN (TB MSN). TB MSN showed maximum antimicrobial activities corresponding to 5.03 and 5.56 log CFU ml^?1 reductions against P. aeruginosa and S. aureus, respectively, whereas samples treated with TB alone showed 2.36 and 2.66 log CFU ml^?1 reductions. Anti-biofilm studies confirmed that TB MSN effectively inhibits biofilm formation and production of extracellular polymeric substances by P. aeruginosa and S. aureus.     

The role of salicylic acid and carrot embryogenic callus extracts in somatic embryogenesis of naked oat (Avena nuda)

Enhanced somatic embryogenesis and plant regeneration have been obtained using young leaf bases of naked oat (Avena nuda) as explants by including salicylic acid (SA) and carrot embryogenic callus extracts (CECE) in media. A 5- and 4-fold improvement was achieved in somatic embryogenesis and plant regeneration on the corresponding media supplemented with 0.5 mM SA and CECE as compared to control, respectively. Some physiological and biochemical changes were assayed in both embryogenic callus (EC) and non-embryogenic callus (NEC). The results indicated that superoxide dismutase activity was stimulated and catalases and ascorbate peroxidase activities were inhibited, while the O₂ ^- (superoxide anion) content was reduced and the hydrogen peroxide level was promoted in EC compared with NEC. Reduced malondialdehyde content and relative electrolyte leakage were also detected in EC.     

Identification of NCF2/p67phox as a novel p53 target gene

Analysis of microarrays performed in p53-, TAp63α- and ΔNp63α-inducible SaOs-2 cell lines allowed the identification of NCF2 mRNA upregulation in response to p53 induction. NCF2 gene encodes for p67phox, the cytosolic subunit of the NADPH oxidase enzyme complex. The recruitment of p67phox to the cell membrane causes the activation of the NADPH oxidase complex followed by the generation of NADP+ and superoxide from molecular oxygen. The presence of three putative p53 binding sites on the NCF2 promoter was predicted, and the subsequent luciferase and chromatin immunoprecipitation assays showed the activation of NCF2 promoter by p53 and its direct binding in vivo to at least one of the sites, thus confirming the hypothesis. NCF2 upregulation was also confirmed by real-time PCR in several cell lines after p53 activation. NCF2 knockdown by siRNA results in a significant reduction of ROS production and stimulates cell death, suggesting a protective function of Nox2-generated ROS in cells against apoptosis. These results provide insight into the redox-sensitive signaling mechanism that mediates cell survival involving p53 and its novel target NCF2/p67phox.     

Ferritin enhances production of DNA strand breaks by 6-hydroxydopamine,ascorbic acid and H2O2 in CCC PM2 bacteriophage DNA

Damage of CCC PM2 DNA by 6-hydroxydopamine (6-OHDA) and ascorbic acid (AA), compounds that are both able to release iron from ferritin, was significantly enhanced in the presence of ferritin. H₂O₂, a product of 6-OHDA autoxidation, did not induce DNA strand breaks in the absence of ferritin and only to a minor extent in the presence of ferritin. DNA damage by 6-OHDA and AA could be reduced by the hydroxyl radical scavenger mannitol, the iron chelator desferrioxamine, and, partly, by a combination of superoxide dismutase and catalase. These inhibitory effects were clearly less pronounced in the presence of ferritin. Ferritin obviously played an important role as a source of iron in the pro-oxidative processes of 6-OHDA and AA. These features might be of importance in cancer therapy since many tumor cells contain elevated ferritin levels.     

Over-expression of Trxo1 increases the viability of tobacco BY-2 cells under H2O2 treatment

Background and Aims Reactive oxygen species (ROS), especially hydrogen peroxide, play a critical role in the regulation of plant development and in the induction of plant defence responses during stress adaptation, as well as in plant cell death. The antioxidant system is responsible for controlling ROS levels in these processes but redox homeostasis is also a key factor in plant cell metabolism under normal and stress situations. Thioredoxins (Trxs) are ubiquitous small proteins found in different cell compartments, including mitochondria and nuclei (Trxo1), and are involved in the regulation of target proteins through reduction of disulphide bonds, although their role under oxidative stress has been less well studied. This study describes over-expression of a Trxo1 for the first time, using a cell-culture model subjected to an oxidative treatment provoked by H₂O₂.Methods Control and over-expressing PsTrxo1 tobacco (Nicotiana tabacum) BY-2 cells were treated with 35 mm H₂O₂ and the effects were analysed by studying the growth dynamics of the cultures together with oxidative stress parameters, as well as several components of the antioxidant systems involved in the metabolism of H₂O₂. Analysis of different hallmarks of programmed cell death was also carried out.Key Results Over-expression of PsTrxo1 caused significant differences in the response of TBY-2 cells to high concentrations of H₂O₂, namely higher and maintained viability in over-expressing cells, whilst the control line presented a severe decrease in viability and marked indications of oxidative stress, with generalized cell death after 3 d of treatment. In over-expressing cells, an increase in catalase activity, decreases in H₂O₂ and nitric oxide contents and maintenance of the glutathione redox state were observed.Conclusions A decreased content of endogenous H₂O₂ may be responsible in part for the delayed cell death found in over-expressing cells, in which changes in oxidative parameters and antioxidants were less extended after the oxidative treatment. It is concluded that PsTrxo1 transformation protects TBY-2 cells from exogenous H₂O₂, thus increasing their viability via a process in which not only antioxidants but also Trxo1 seem to be involved.     

Cloning and characterization of a 2-cys peroxiredoxin in the pine wood nematode, Bursaphelenchus xylophilus, a putative genetic factor facilitating the infestation

The pine wood nematode, Bursaphelenchus xylophilus, is an invasive plant parasitic nematode and a worldwide quarantine pest. An indigenous species in North America and the causal agent of pine wilt disease, B. xylophilus has devastated pine production in Southeastern Asia including Japan, China, and Korea since its initial introduction in the early 1900s. The reactive oxygen species (ROS) is the first line of defense utilized by host plants against parasites, while nematodes, counteractively, employ antioxidants to facilitate their infection. Peroxiredoxins (Prxs) are a large class of antioxidants recently found in a wide variety of organisms. In this report, a gene encoding a novel 2-cysteine peroxiredoxin protein in B. xylophilus was cloned and characterized. The 2-cysteine peroxiredoxin in B. xylophilus (herein refers to as "BxPrx") is highly conserved in comparison to 2-cysteine peroxiredoxins (Prx2s) in other nematodes, which have two conserved cysteine amino acids (Cp and Cr), a threonine-cysteine-arginine catalytic triad, and two signature motifs (GGLG and YF) sensitive to hydrogen peroxide. In silico assembly of BxPrx tertiary structure reveals the spatial configuration of these conserved domains and the simulated BxPrx 3-dimensional structure is congruent with its presumed redox functions. Although no signal peptide was identified, BxPrx was abundantly expressed and secreted under the B. xylophilus cuticle. Upon further analysis of this leader-less peptide, a single transmembrane α-helix composed of 23 consecutive hydrophobic amino acids was found in the primary structure of BxPrx. This transmembrane region and/or readily available ATP binding cassette transporters may facilitate the transport of non-classical BxPrx across the cell membrane. Recombinant BxPrx showed peroxidase activity in vitro reducing hydrogen peroxide using glutathione as the electron donor. The combined results from gene discovery, protein expression and distribution profiling (especially the "surprising" presence under the nematode cuticle), and recombinant antioxidant activity suggest that BxPrx plays a key role in combating the oxidative burst engineered by the ROS defense system in host plants during the infection process. In summary, BxPrx is a genetic factor potentially facilitating B. xylophilus infestation.     

Sec-containing TrxR1 is essential for self-sufficiency of cells by control of glucose-derived H2O2

It is commonly recognized that diabetic complications involve increased oxidative stress directly triggered by hyperglycemia. The most important cellular protective systems against such oxidative stress have yet remained unclear. Here we show that the selenoprotein thioredoxin reductase 1 (TrxR1), encoded by the Txnrd1 gene, is an essential enzyme for such protection. Individually grown Txnrd1 knockout (Txnrd1^−/−) mouse embryonic fibroblasts (MEFs) underwent massive cell death directly linked to glucose-induced H₂O₂ production. This death and excessive H₂O₂ levels could be reverted by reconstituted expression of selenocysteine (Sec)-containing TrxR1, but not by expression of Sec-devoid variants of the enzyme. Our results show that Sec-containing TrxR1 is absolutely required for self-sufficient growth of MEFs under high-glucose conditions, owing to an essential importance of this enzyme for elimination of glucose-derived H₂O₂. To our knowledge, this is the first time a strict Sec-dependent function of TrxR1 has been identified as being essential for mammalian cells.     

Comparative study of the formation of oxidative damage marker 8-hydroxy-2'-deoxyguanosine (8-OHdG) adduct from the nucleoside 2'-deoxyguanosine by transition metals and suspensions of particulate matter in relation to metal content and redox reactivity

An association between exposure to ambient particulate matter (PM) and increased incidence of mortality and morbidity due to lung cancer and cardiovascular diseases has been demonstrated by recent epidemiological studies. Reactive oxygen species (ROS), especially hydroxyl radicals, generated by PM, have been suggested by many studies as an important factor in the oxidative damage of DNA by PM. The purpose of this study was to characterize quantitatively hydroxyl radical generation by various transition metals in the presence of H₂O₂ in aqueous buffer solution (pH 7.4) and hydroxylation of 2'-deoxyguanosine (dG) to 8-hydroxy-2'-deoxyguanosine (8-OHdG) under similar conditions. The order of metals' redox reactivity and hydroxyl radical production was Fe(II), V(IV), Cu(I), Cr(III), Ni(II), Co(II), Pb(II), Cd(II). Then, we investigated the generation of hydroxyl radicals in the presence of H₂O₂ by various airborne PM samples, such as total suspended particulate (TSP), PM₁₀, PM_2.5 (PM with aerodynamic diameter 10 and 2.5 μm), diesel exhaust particles (DEP), gasoline exhaust particles (GEP) and woodsmoke soot under the same conditions. When suspensions of PMs were incubated with H₂O₂ and dG at pH 7.4, all particles induced hydroxylation of dG and formation of 8-OHdG in a dose-dependent increase. Our findings demonstrated that PM's hydroxyl radical (HO√) generating ability and subsequent dG hydroxylation is associated with the concentration of water-soluble metals, especially Fe and V and other redox or ionizable transition metals and not their total metal content, or insoluble metal oxides, via a Fenton-driven reaction of H₂O₂ with metals. Additionally, we observed, by Electron paramagnetic resonance (EPR), that PM suspensions in the presence of H₂O₂ generated radical species with dG, which were spin-trapped by 2-methyl-2-nitroso-propane (MNP).     

Up-regulation of antioxidant enzymes and coenzyme Q10 in a human oral cancer cell line with acquired bleomycin resistance

Abstract

Bleomycin (BLM) is an anti-cancer drug that can induce formation of reactive oxygen species (ROS). To investigate the association between up-regulation of antioxidant enzymes and coenzyme Q₁₀ (CoQ₁₀) in acquired BLM resistance, one BLM-resistant clone, SBLM24 clone, was selected from a human oral cancer cell line, SCC61 clone. The BLM resistance of SBLM24 clone relative to a sub-clone of SCC61b cells was confirmed by analysis of clonogenic ability and cell cycle arrest. CoQ₁₀ levels and levels of Mn superoxide dismutase, glutathione peroxidase 1, catalase and thioredoxin reductase 1 were augmented in SBLM24 clone although there was also a mild increase in the expression of BLM hydrolase. Suppression of CoQ₁₀ levels by 4-aminobenzoate sensitized BLM-induced cytotoxicity. The results of suppression on enhanced ROS production by BLM and the cross-resistance to hydrogen peroxide in SBLM24 clone further demonstrated the development of adaptation to oxidative stress during the formation of acquired BLM resistance.     

Expression of ascorbate peroxidase and glutathione reductase in roots of rice seedlings in response to NaCl and H2O2

The accumulation of H2O2 by NaCl was observed in the roots of rice seedlings. Treatment with NaCl caused an increase in the activities of ascorbate peroxidase (APX) and glutathione reductase (GR) and the expression of OsAPX and OsGR in rice roots. Exogenously applied H2O2 also enhanced the activities of APX and GR and the expression of OsAPX and OsGR in rice roots. The accumulation of H2O2 in rice roots in response to NaCl was inhibited by the NADPH oxidase inhibitors, diphenyleneiodonium chloride (DPI) and imidazole (IMD). However, DPI, IMD, and dimethylthiourea, a H2O2 trap, did not reduce NaCl-enhanced activities of APX and GR and expression of OsAPX and OsGR. It appears that H2O2 is not involved in the regulation of NaCl-induced APX and GR activities and OsAPX and OsGR expression in rice roots.