Decreased reactive oxygen generation during H2O2 decomposition in the presence of samples from human rectal cancer

Reactive oxygen species (ROS) have generated a great deal of interest in the clinical field since experimental studies showed the involvement of these species in carcinogenesis. This paper reports the detection of ROS during the decomposition of H2O2 in the presence of samples obtained from tissues of 16 patients with rectal carcinoma (age 64 +/- 9 years) operated on in the Division of Surgical Oncology of Pomeranian Medical University, Szczecin (Poland). The samples were cut from the middle of the resected tumors and from the colonic mucosa (10 cm distant from the tumor and free of disease); they were processed and the supernatants, representing the soluble fraction, were used for measurements. Various methods for measuring free radical activity of the examined samples were used, such as chemiluminescence, fluorescent probe 2',7'-dichlorodihydrofluorescein, spin trap 5,5-dimethyl-pyrroline-1-oxide and EPR, the spectrophotometrically examined formation of diformazan during reduction of the p-nitroblue tetrazolium salt, and bleaching of p-nitrosodimethylalanine. A statistically significant difference (P < 0.001) was noticed in mean chemiluminescence +/- standard error of the mean in the presence of the tumor samples (42.6 +/- 7.3) in comparison to the control samples (234.6 +/- 36.0). Significantly decreased generation of ROS from the decomposition of H2O2 in the presence of the tumor samples in comparison to the control samples was also observed when the above-mentioned methods were used. Tumor samples had significantly lower superoxide dismutase activity (33 +/- 4 U/mg protein) than controls (93 +/- 14 U/mg, P < 0.001), which should contribute to a lower capacity of endogenous H2O2 production and therefore less ROS generation upon H2O2 decomposition. We conclude that the tested samples have different redox properties; this supports a possible role of ROS activity during carcinogenesis. Moreover, we propose a new, simple, and sensitive chemiluminescent method, which might be effective in sample differentiation.     

Regulation of brain mitochondrial H2O2 production by membrane potential and NAD(P)H redox state

Mitochondrial production of reactive oxygen species (ROS) at Complex I of the electron transport chain is implicated in the etiology of neural cell death in acute and chronic neurodegenerative disorders. However, little is known regarding the regulation of mitochondrial ROS production by NADH-linked respiratory substrates under physiologically realistic conditions in the absence of respiratory chain inhibitors. This study used Amplex Red fluorescence measurements of H2O2 to test the hypothesis that ROS production by isolated brain mitochondria is regulated by membrane potential (DeltaPsi) and NAD(P)H redox state. DeltaPsi was monitored by following the medium concentration of the lipophilic cation tetraphenylphosphonium with a selective electrode. NAD(P)H autofluorescence was used to monitor NAD(P)H redox state. While the rate of H2O2 production was closely related to DeltaPsi and the level of NAD(P)H reduction at high values of DeltaPsi, 30% of the maximal rate of H2O2 formation was still observed in the presence of uncoupler (p-trifluoromethoxycarbonylcyanide phenylhydrazone) concentrations that provided for maximum depolarization of DeltaPsi and oxidation of NAD(P)H. Our findings indicate that ROS production by mitochondria oxidizing physiological NADH-dependent substrates is regulated by DeltaPsi and by the NAD(P)H redox state over ranges consistent with those that exist at different levels of cellular energy demand.     

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.     

Epigallocatechin‐3‐gallate induces mesothelioma cell death via H2O2−dependent T‐type Ca2+ channel opening

Malignant mesothelioma (MMe) is a highly aggressive, lethal tumour requiring the development of more effective therapies. The green tea polyphenol epigallocathechin‐3‐gallate (EGCG) inhibits the growth of many types of cancer cells. We found that EGCG is selectively cytotoxic to MMe cells with respect to normal mesothelial cells. MMe cell viability was inhibited by predominant induction of apoptosis at lower doses and necrosis at higher doses. EGCG elicited H₂O₂ release in cell cultures, and exogenous catalase (CAT) abrogated EGCG‐induced cytotoxicity, apoptosis and necrosis. Confocal imaging of fluo 3‐loaded, EGCG‐exposed MMe cells showed significant [Ca²⁺]_i rise, prevented by CAT, dithiothreitol or the T‐type Ca²⁺ channel blockers mibefradil and NiCl₂. Cell loading with dihydrorhodamine 123 revealed EGCG‐induced ROS production, prevented by CAT, mibefradil or the Ca²⁺ chelator BAPTA‐AM. Direct exposure of cells to H₂O₂ produced similar effects on Ca²⁺ and ROS, and these effects were prevented by the same inhibitors. Sensitivity of REN cells to EGCG was correlated with higher expression of Ca_v3.2 T‐type Ca²⁺ channels in these cells, compared to normal mesothelium. Also, Ca_v3.2 siRNA on MMe cells reduced in vitro EGCG cytotoxicity and abated apoptosis and necrosis. Intriguingly, Ca_v3.2 expression was observed in malignant pleural mesothelioma biopsies from patients, but not in normal pleura. In conclusion, data showed the expression of T‐type Ca²⁺ channels in MMe tissue and their role in EGCG selective cytotoxicity to MMe cells, suggesting the possible use of these channels as a novel MMe pharmacological target.     

Mitogen-activated protein kinases modulate H(2)O(2)-induced apoptosis in primary rat alveolar epithelial cells

Increasing evidence suggests a role for apoptosis in the maintenance of the alveolar epithelium under normal and pathological conditions. However, the signaling pathways modulating alveolar type II (ATII) cell apoptosis remain poorly defined. Here we investigated the role of MAPKs as modulators of oxidant-mediated ATII cell apoptosis using in vitro models of H(2)O(2)-stress. H(2)O(2), delivered either as a bolus or as a flux, lead to time- and concentration-dependent increases in ATII cells apoptosis. Increased apoptosis in primary rat ATII cells was detected at H(2)O(2) concentrations and production rates in the physiological range (1 microM) and peaked at 100 microM H(2)O(2). Immortalized rat lung epithelial cells (RLE), in contrast, required millimolar concentration of H(2)O(2) for maximal responses. H(2)O(2)-induced apoptosis was preceded by rapid activation of all three classes of mitogen-activated protein kinases (MAPKs): ERK, JNK, and p38. Specific inhibition of JNK using antisense oligonucleotides and ERK and p38 using PD98059 or SB202190, respectively, indicated a pro-apoptotic role for JNK pathway and an anti-apoptotic role for ERK- and p38-initiated signaling events. Our data show that the balance between the activation of JNK, ERK, and p38 is a critical determinant of cell fate, suggesting that pharmacological interventions on the MAPK pathways may be useful in the treatment of oxidant-related lung injury.     

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.     

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.     

Ag-SiO_2核壳型纳米粒的制备及其抗菌作用

利用抗坏血酸对AgNO3进行还原,生成银纳米粒核心,并通过正硅酸四乙酯的水解与聚合反应获得SiO2介孔外壳,制备平均粒径约为92.9 nm的Ag-SiO2核-壳型纳米粒。Ag-SiO2纳米粒可以显著地抑制香石竹镰刀菌的生长,最小抑菌质量浓度为4μg/mL,并可抑制香石竹镰刀菌菌丝生长和孢子分生。Ag-SiO2纳米粒处理2~4 h后,菌丝体的过氧化氢酶、总超氧化物歧化酶、过氧化物酶活力增强,提示Ag-SiO2纳米粒抗菌机制和活性氧诱导相关。     

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.     

Effects and mechanisms of H(2)O(2) on production of dicarboxylic acid.

The system of producing long chain dicarboxylic acid (DCA) by Candida tropicalis is an aerobic and viscous fermentation system. A method to overcome the gas-liquid transport resistance and to increase oxygen supply is by adding hydrogen peroxide (H(2)O(2)) to the fermentation system. Here we report that the H(2)O(2) not only can enhance the oxygen supply but also change the metabolism by inducing cytochrome P450, the key enzyme of a, o-oxidation. When C. tropicalis was cultivated in a 3-L bioreactor using the combination of aeration and H(2)O(2) feeding, DCA production rates increased by about 10% after a short period of decrease at the beginning. Furthermore, the experiments showed that the maximum activities of P450 could be induced at 2 mM H(2)O(2), and the inducible mechanisms are also discussed. Moreover, we suggest that alkane might be oxidized through the "peroxide shunt pathway" when H(2)O(2) is present. By adding H(2)O(2), the DCA yield in a 22-L bioreactor could increase by 25.3% and reach 153.9 g/L.     

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.     

Activation of γ-Aminobutyrate Production by Chloroplastic H2O2 Is Associated with the Oxidative Stress Response

A series of N-substituted aryl and alkyl carbamates (RNHCOOR′; R: aryl, alkyl; R′: aryl, alkyl) was prepared and screened for inhibitory activity toward the germination of oat seeds. The activity of each compound was compared with that of chlorpropham (isopropyl 3-chlorocarbanilate). Some of the synthetic carbamates possessing the N-(phenylthio)methyl group, PhSCH₂NHCOOR´, showed inhibitory activity close or comparable to that of chlorpropham.     

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.     

A novel role for cyanide in the control of cucumber (Cucumis sativus L.) seedlings response to environmental stress

The effects of potassium cyanide (KCN) pretreatment on the response of cucumber (Cucumis sativus L.) plants to salt, polyethylene glycol (PEG) and cold stress were investigated in the present study. Here, we found that KCN pretreatment improved cucumber seedlings tolerance to stress conditions with maximum efficiency at a concentration of 20 µM. The results showed that pretreatment with 20 µM KCN alleviated stress‐induced oxidative damage in plant cells and clearly induced the activity of alternative oxidase (AOX) and the ethylene production. Furthermore, the structures of thylakoids and mitochondria in the KCN‐pretreated seedlings were less damaged by the stress conditions, which maintained higher total chlorophyll content, photosynthetic rate and photosystem II (PSII) proteins levels than the control. Importantly, the addition of the AOX inhibitor salicylhydroxamic acid (1 mm ; SHAM) decreased plant resistance to environmental stress and even compromised the cyanide (CN)‐enhanced stress tolerance. Therefore, our findings provide a novel role of CN in plant against environmental stress and indicate that the CN‐enhanced AOX might contribute to the reactive oxygen species (ROS) scavenging and the protection of photosystem by maintaining energy charge homoeostasis from chloroplast to mitochondria.     

Percentage of phagocytosis,production of O2·−, H2O2 and NO,and antioxidant enzyme activities of rat neutrophils in culture

Changes in the integrity, ultrastructure, phagocytosis capacity, and production of H₂O₂, O₂^{· −}and NO₂⁻ were evaluated in cultured neutrophils. The activities of the antioxidant enzymes (catalase—CAT, superoxide dismutase—SOD and glutathione-dependent peroxidase—GSH-Px) were measured under similar conditions. The integrity of the cells remained unchanged up to 18 h. After 24 h, the number of viable cells in culture dropped by 16 per cent. The percentage of viable cells in culture was of 72 per cent even after 72 h. An ultrastructural analysis of the cells was carried out after 3, 6, 12, 24, 48, and 72 h in culture. Neutrophils started developing morphologic changes after 24 h: decreased cell volume, abundant vacuoles (mainly around the nucleus), and also the presence of autophagic vacuoles. This period was then chosen for the study of neutrophil function and antioxidant enzyme activities. Neutrophils cultured for 24 h presented reduced phagocytosis capacity. The rates of production of H₂O₂ and O₂^{· −} remained unchanged after 24 h in culture. Concomitantly, these cells were also able to produce NO in significant amounts. The production of O₂^·− in response to PMA stimulus was lowered in 24-h cultured cells. Possibly, the production of oxygen and nitrogen reactive species accomplished with a decrease in the activities of CAT and GSH-Px play a key role for the process of apoptosis which takes place in neutrophils under these conditions. © 1998 John Wiley & Sons, Ltd.     

H2O2‐ and pH‐sensitive CdTe quantum dots as fluorescence probes for the detection of glucose

A novel fluorescence assay system for glucose was developed with thioglycollic acid (TGA)‐capped CdTe quantum dots (QDs) as probes. The luminescence quantum yield of the TGA‐capped CdTe QDs was highly sensitive to H₂O₂ and pH. In the presence of glucose oxidase, glucose is oxidized to yield, gluconic acid and H₂O₂. H₂O₂ and H⁺ (dissociated from gluconic acid) intensively quenched the fluorescence of QDs. The experimental results showed that the quenched fluorescence was proportional to the glucose concentration within the range of 0.01–5.0 mm under optimized experimental conditions. Compared with most of the existing methods, this newly developed system possesses many advantages, including simplicity, low cost, high flexibility, and good sensitivity. Furthermore, no complicated chemical modification of QDs and enzyme immobilization was needed in this system. Copyright © 2012 John Wiley & Sons, Ltd.     

ROS production and protein oxidation as a novel mechanism for seed dormancy alleviation

At harvest, sunflower (Helianthus annuus L.) seeds are dormant and unable to germinate at temperatures below 15 degrees C. Seed storage in the dry state, known as after-ripening, is associated with an alleviation of embryonic dormancy allowing subsequent germination at suboptimal temperatures. To identify the process by which dormancy is broken during after-ripening, we focused on the role of reactive oxygen species (ROS) in this phenomenon. After-ripening entailed a progressive accumulation of ROS, namely superoxide anions and hydrogen peroxide, in cells of embryonic axes. This accumulation, which was investigated at the cellular level by electron microscopy, occurred concomitantly with lipid peroxidation and oxidation (carbonylation) of specific embryo proteins. Incubation of dormant seeds for 3 h in the presence of hydrogen cyanide (a compound that breaks dormancy) or methylviologen (a ROS-generating compound) also released dormancy and caused the oxidation of a specific set of embryo proteins. From these observations, we propose a novel mechanism for seed dormancy alleviation. This mechanism involves ROS production and targeted changes in protein carbonylation patterns.