Cell inactivation studies on yeast cells under euoxic and hypoxic condition using electron beam from microtron accelerator

In the case of sparsely ionizing radiation such as electron, the dose rate and the pattern of energy deposition of the radiation are the important physical factors which can affect the amount of damage in living cells. In the present study, the differences in the cell survival efficiency and dose rate effect in diploid yeast strains Saccharomyces cerevisiae X2180 and Saccharomyces cerevisiae D7 under euoxic and hypoxic condition have been quantified. Irradiation was carried out using 8 MeV pulsed electron beam from Microtron accelerator. The dose per pulse and pulse width of the beam used was 0.6 Gy and 2.3 μs, respectively, which correspond to an instantaneous dose rate of 2.6 × 10⁵ Gy s⁻¹. For survival studies doses were delivered at a rate of 50 pulses per second (an average dose rate of 1,800 Gy s⁻¹). Fricke and alanine dosimeters were used to measure the dose delivered to the sample. A significant difference in the dose response has been observed under euoxic and hypoxic condition. Dose rate effect has been studied by changing the pulse repetition rate of the Microtron and the dose rate used was from 180 to 1800 Gy min⁻¹. A significant dose rate effect was observed under euoxic condition for Saccharomyces cerevisiae X2180 but the same was absent under hypoxic condition. The dose rate effect was absent for Saccharomyces cerevisiae D7 under both irradiation condition. The survival curves are found to be sigmoidal in shape under both condition but with a wider shoulder under hypoxic condition. The D₀ value and the Oxygen Enhancement Ratio (OER) at that point have been derived.     

Production of reactive oxygen species in endothelial cells under different pulsatile shear stresses and glucose concentrations

A hemodynamic Lab-on-a-chip system was developed in this study. This system has two unique features: (1) it consists of a microfluidic network with an array of endothelial cell seeding sites for testing them under multiple conditions, and (2) the flow rate and the frequency of the culture medium in the microchannel are controlled by a pulsation free pump to mimic the flow profile of the blood in the blood vessel under different physiological conditions. The investigated physiological conditions were: (1) the resting condition in a normal shear stress of 15 dyne cm(-2) with a normal heart rate of 70 bpm, (2) an exhaustive exercise condition with a high shear stress of 30 dyne cm(-2) and a fast heart rate of 140 bpm, and (3) a constant high shear stress of 30 dyne cm(-2). Two chemical conditions were investigated (10 mM and 20 mM glucose) to mimic hyperglycemic conditions in diabetes patients. The effects of various shear stresses either alone or in combination with different glucose concentrations on endothelial cells were examined using the developed hemodynamic Lab-on-a-chip system by assessing two parameters. One is the intracellular level of reactive oxygen species (ROS) determined by a fluorescent probe, H(2)DCFDA. Another is the mitochondrial morphology revealed with a fluorescent dye, MitoTracker Green FM. The results showed that ROS level was elevated nearly 4-fold after 60 min of exhaustive exercise. We found that the pulsatile nature of the fluid was the determination factor for causing ROS generation in the cells as almost no increase of ROS was detected in the constant shear stress condition. Similarly, much higher level of ROS was detected when 10 mM glucose was applied to the cells under normal or high pulsatile shear stresses compared with under a static condition. These results suggest that it is necessary to use pulsatile shear stress to represent the physiological conditions of the blood flow, and demonstrate the advantage of utilizing this newly developed hemodynamic Lab-on-a-chip system over the conventional non-pulsatile system in the future shear stress related studies.     

Trichosanthin induced calcium-dependent generation of reactive oxygen species in human choriocarcinoma cells

The type-I ribosome-inactivating protein trichosanthin (TCS) has a broad spectrum of biological and pharmacological activities, including abortifacient, anti-tumor and anti-HIV. We found for the first time that TCS induced the generation of reactive oxygen species (ROS) in human choriocarcinoma cells (JAR cells) at the level of the single cell by using the fluorescent probe 2',7'-dichlorofluorescein diacetate with confocal laser scanning microscopy. TCS-induced ROS formation was shown to be dependent on the presence of extracellular Ca2+ and was further reduced when cytosolic Ca2+ was chelated by BAPTA-AM. The production of ROS increased rapidly after the application of TCS, which paralleled TCS-induced increase in intracellular calcium monitored using fluo 3-AM. Simultaneous observation of the nuclear morphological changes via two-photon laser scanning microscopy and production of ROS via confocal laser scanning microscopy revealed that ROS were involved in the apoptosis of JAR cells. The contribution of ROS was confirmed by experiments in which the antioxidant alpha-tocopherol prevented TCS-induced ROS formation and cell death. The finding that TCS induced calcium-dependent generation of ROS in JAR cells and that ROS were involved in the apoptosis of JAR cells might provide new insight into the anti-tumor and anti-HIV mechanism of TCS.     

Lutein, zeaxanthin and astaxanthin protect against DNA damage in SK-N-SH human neuroblastoma cells induced by reactive nitrogen species

The purpose of this study was to evaluate the ability of the predominant carotenoids (lutein and zeaxanthin) of the macular pigment of the human retina, to protect SK-N-SH human neuroblastoma cells against DNA damage induced by different RNOS donors. Although astaxanthin has never been isolated from the human eye, it was included in this study because its structure is very close to that of lutein and zeaxanthin and because it affords protection from UV-light. DNA damage was induced by GSNO-MEE, a nitric oxide donor, by Na(2)N(2)O(3), a nitroxyl anion donor and by SIN-1, a peroxynitrite-generating agent. DNA damage was assessed using the comet assay, a rapid and sensitive single cell gel electrophoresis technique able to detect primary DNA damage in individual cells. The tail moment parameter was used as an index of DNA damage. The values of tail moment increased in all the samples incubated with the RNOS donors, indicating DNA impairment. Data obtained show that the ability of zeaxanthin, lutein, and astaxanthin to reduce the DNA damage depends on the type of RNOS donor and the carotenoid concentration used. All the carotenoids studied were capable of protecting against DNA damage in neuroblastoma cells when the cells were exposed to GSNO-MEE. However, a different behaviour was present when the other two RNOS donors were used. The presence of a carotenoid alone (without an RNOS donor) did not cause DNA damage. Spectrophotometric studies showed that the order with which tested carotenoids reacted with RNOS was not always in agreement with the DNA protection results. The data from this study provides additional information on the activities of the macular pigment carotenoids of the human retina.     

Secondary reactive oxygen species extend the range of photosensitization effects in cells: DNA damage produced via initial membrane photosensitization

The type-II photosensitization process is mediated by the formation of singlet oxygen (O2[1deltag]). The short lifetime of this species dictates that chemical reactions with biological substrates can only occur when O2(1deltag) is in very close proximity to the photosensitizer itself. In this study, deuteroporphyrin, a type-II, membrane-localized photosensitizer, was used to generate O2(1deltag) in human lymphoblast WTK-1 cells, and the range of influence was determined by a variety of biological assays. Surprisingly, the initial membrane-confined events were shown, by comet assay, to induce DNA damage in these cells. DNA damage was inhibited both by membrane-localized (alpha-tocopherol acetate) and by cytoplasmic (trolox) free radical scavengers. Comet formation also was inhibited by treatment at low temperature. DNA fragmentation was not influenced by treatment with the pan-caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone, showing that apoptosis was not responsible for fragmentation. Taken together, these results show that primary photosensitization reactions involving O2(1deltag), even when tightly confined in extranuclear locations, leads to the production of secondary reactive oxygen species, probably as a result of lipid peroxidation, that can act at greater distances from the photosensitizer itself. These experiments were carried out under conditions where cell survival was significant and raise questions regarding DNA damage and mutagenesis pathways, even when extranuclear O2(1deltag)-generating compounds are used.     

Involvement of reactive oxygen species and calcium in photo-induced membrane damage in HeLa cells by a bis-methanophosphonate fullerene

Photo-excited bioactivities of fullerene derivatives are attracting much attention. In this report, a bis-methanophosphonate fullerene (BMPF) and the other two fullerene derivatives, a bis-malonic acid fullerene (BMAF) and a fullerol were incubated with HeLa cells and irradiated with a green light emitted from a mercury lamp on a fluorescent microscopy. By using DNA fluorescent probe propidium iodide staining method, damage towards cell membrane could be detected when cells were treated by irradiation altogether with BMPF or BMAF at a low concentration (4 μM), and the damage was dose-dependent. The activity of BMPF was much higher than that of BMAF, while fullerol had no effects under the same condition. It was also revealed that different kinds of reactive oxygen species (ROS) correlated to BMPF and BMAF. Additionally, presence of extracellular calcium could promote the activities of both derivatives, while removal of extracellular calcium could not abort their membrane-damaged activities. These results indicated that ROS and calcium were involved in the photosensitization of fullerene derivatives, and BMPF was a superior photosensitizer which would find potential application in biomedical field.     

Production of reactive oxygen species on photolysis of dilute aqueous quinone solutions

We have examined the generation of the reactive oxygen species (ROS) superoxide and hydrogen peroxide (H2O2) by irradiation of dilute aqueous solutions of disodium anthraquinone-2-6-disulfonate (AQDS) with simulated sunlight. Irradiating a solution of AQDS in 2 mM NaHCO3 and 0.01 M NaCl produced superoxide and H2O2 at nanomolar concentrations. Experiments in which initial concentrations of dioxygen, H2O2, the superoxide radical trap nitroblue tetrazolium and the electron donor dimethyl sulfoxide were varied suggested that the interaction of solvent water with photo-excited quinone moieties produces dioxygen-reducing radicals, and that these are the primary source of ROS in the system. A kinetic model for ROS production is proposed based on our experimental data.     

Involvement of reactive oxygen species in the UV-B damage to the cyanobacterium Anabaena sp

Reactive oxygen species (ROS) are involved the damage of living organisms under environmental stress including UV radiation. Cyanobacteria, photoautotrophic prokaryotic organisms, also suffer from increasing UV-B due to the depletion of the stratospheric ozone layer. The increased UV-B induces the production of ROS in vivo detected by using the ROS-sensitive probe 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA). Ascorbic acid and N-acetyl-L-cysteine (NAC) scavenged ROS effectively, while alpha-tocopherol acetate or pyrrolidine dithiocarbamate (PDTC) did not. The presence of rose bengal and hypocrellin A increased the ROS level by photodynamic action in the visible light. The presence of the herbicide, 3-(3,4-dichlorophenyl)-1,1-dimethyl urea (DCMU), increased ROS production slightly, and ROS formation was greatly enhanced by the addition of methyl viologen due to the fact that this redox system diverts electrons from PSI to oxygen and thus forms ROS. UV-B induces ROS generation by photodynamic action and inhibition of the electron transport by damaging the electron receptors or enzymes associated with the electron transport chain during photosynthesis.     

Characterization of reactive oxygen species generated by protoporphyrin IX under X-ray irradiation

As a possible radiosensitizer candidate having biological compatibility, oncotropic property, and X-ray activation capability, contribution of protoporphyrin IX (PpIX) to enhanced generation of reactive oxygen species (ROS) under X-ray and UV irradiations were examined. To identify the kinds of ROS, 2-[6-(4-amino)phenoxy-3H-xanthen-3-on-9-yl] benzoic acid (APF) and dihydroethidium (DHE) were used together with ethanol as a hydroxyl radical (OH) quencher. All of the three species of our interest (OH, superoxide radical (O₂⁻), and singlet oxygen (¹O₂)) were enhanced by PpIX under X-ray and UV irradiations in addition to those by radiolysis and photolysis. Its enhancement factors exceeded 1.7 depending on the concentrations of PpIX from 1.5 to 15.0 μg/ml.     

Simultaneous determination of glutathione and reactive oxygen species in individual cells by microchip electrophoresis

A microchip electrophoresis method was developed for simultaneous determination of reactive oxygen species (ROS) and reduced glutathione (GSH) in the individual erythrocyte cell. In this method, cell sampling, single-cell loading, docking, lysing, and capillary electrophoretic separation with LIF detection were integrated on a microfluidic chip with crossed channels. ROS was labeled with dihydrorhodamine 123 in the intact cell, while GSH was on-chip labeled with 2,3-naphthalene-dicarboxaldehyde, which was included in the separation medium. On-chip electrical lysis, characterized by extremely fast disruption of the cellular membrane (<40 ms), was exploited to minimize enzymatic effects on analyte concentrations during the determination. The microfluidic network was optimized to prevent cell leaking from the sample reservoir (S) into separation during the separation phase. The structure of the S was modified to avoid blockage of its outlet by deposited cells. Detection limits of 0.5 and 6.9 amol for ROS and GSH, respectively, were achieved. The average cell throughput was 25 cells/h. The effectiveness of the method was demonstrated in the simultaneous determination of GSH and ROS in individual cells and the variations of cellular GSH and ROS contents in response to external stimuli.     

Nanoelectrodes for intracellular measurements of reactive oxygen and nitrogen species in single living cells

Reactive oxygen and nitrogen species (ROS and RNS) play important roles in various physiological processes (e.g. phagocytosis) and pathological conditions (e.g. cancer). The primary ROS/RNS, viz., hydrogen peroxide, peroxynitrite ion, nitric oxide, and nitrite ion, can be oxidized at different electrode potentials and therefore detected and quantified by electroanalytical techniques. Nanometer-sized electrochemical probes are especially suitable for measuring ROS/RNS in single cells and cellular organelles. In this article, we survey recent advances in the localized measurements of ROS/RNS inside single cells and discuss several methodological issues, including optimization of nanoelectrode geometry, precise positioning of an electrochemical probe inside a cell, and interpretation of electroanalytical data.     

Photogeneration of reactive oxygen species and photoinduced plasmid DNA cleavage by novel synthetic chalcones

This paper describes the synthesis and photodynamic properties of six different chalcone derivatives. Using N,N-dimethyl-4-nitrosoaniline (RNO) bleaching assay, the singlet oxygen generating efficiencies of these chalcones are determined relative to rose bengal (RB). Superoxide dismutase (SOD) inhibitable cytochrome c reduction assay and electron magnetic resonance (EMR) spin trapping techniques are used to determine the superoxide anion radical (O?·?) yield upon photoirradiation. Photoinduced DNA scission studies show that O?·? is involved in the DNA strand break. In addition, antimicrobial activity of these chalcones is also investigated. Structure activity relationship accounts for the difference in the photogeneration of reactive oxygen species (ROS) by these sensitizers. Presence of electron releasing -OCH? groups enhances the photogeneration of ROS. Cyclic voltammetry studies indicate a correlation between enzymatic O?·? generation efficiency and redox potential of chalcones. Both O?·? (Type I) and 1O? (Type II) paths are involved in the photosensitization of chalcones. The LUMO energies obtained by molecular modeling correlate with the one-electron reduction potentials.     

Involvement of reactive oxygen species and reactive nitrogen species in the wound response of Dasycladus vermicularis

We investigated the signaling events involved in the wound response of the marine macroalga Dasycladus vermicularis, finding nitric oxide (NO) production in relation to injury. The addition of exogenous H2O2 to aliquots of injured algae accelerated the kinetics of NO production in the wounded region. Similarly, the addition of an NO donor caused an increase in detectable H2O2 around the site of injury. By wounding or incubating uninjured algae with an NO donor, peroxidase activity was enhanced. Based on the use of selected pharmacological probes, our results indicate that H2O2 production involves the upstream activation of signaling events similar to those observed in the physiology of higher plants.     

Production of reactive oxygen species induced by a new [60]fullerene derivative bearing a tetrazole unit and its possible biological applications

The wide range of physical and chemical properties of modified fullerenes has drawn increasing attention in the past few years. As part of this research, this paper describes the preparation, characterization, and photophysical properties of a new fullerene derivative chemically modified with a tetrazole. The photophysical properties were studied by EPR radical spin-trapping technique and showed that reactive oxygen species (ROS) can be produced through UVA photosensitization. EPR spin-trapping experiments with singlet oxygen (¹O₂) and superoxide (O₂⁻) inhibitors (β-carotene and superoxide dismutase, respectively) revealed also that: (i) the main ROS produced is ¹O₂ and (ii) ¹O₂ is being partially dismutated to O₂⁻. The results suggest that this derivative can be used in biological applications, as for example, in topic photodynamic therapy (PDT) as a photosensitizer.     

Mitochondria-derived reactive oxygen species mediate blue light-induced death of retinal pigment epithelial cells

Throughout the lifetime of an individual, light is focused onto the retina. The resulting photooxidative stress can cause acute or chronic retinal damage. The pathogenesis of age-related macular degeneration (AMD), the leading cause of legal blindness in the developed world, involves oxidative stress and death of the retinal pigment epithelium (RPE) followed by death of the overlying photoreceptors. Evidence suggests that damage due to exposure to light plays a role in AMD and other age-related eye diseases. In this work a system for light-induced damage and death of the RPE, based on the human ARPE-19 cell line, was used. Induction of mitochondria-derived reactive oxygen species (ROS) is shown to play a critical role in the death of cells exposed to short-wavelength blue light (425 +/- 20 nm). ROS and cell death are blocked either by inhibiting the mitochondrial electron transport chain or by mitochondria-specific antioxidants. These results show that mitochondria are an important source of toxic oxygen radicals in blue light-exposed RPE cells and may indicate new approaches for treating AMD using mitochondria-targeted antioxidants.     

An EPR characterisation of stable and transient reactive oxygen species formed under radiative and non-radiative conditions

Research on Chemical Intermediates - Electron paramagnetic resonance (EPR) spectroscopy is the ideal method of choice when detecting and studying the wide variety of paramagnetic oxygen-centred...     

Linifanib induces apoptosis in human ovarian cancer cells via activation of FOXO3 and reactive oxygen species

Linifanib is known as an inhibitor of receptor tyrosine kinase. Even though it has been widely recognized as efficient inhibitor of receptor tyrosine kinases, anti-carcinogenic effect has not been investigated enough in ovarian cancer. In this study, we investigated the anti-cancer effect of linifanib on human ovary cancer SKOV3 cells. WST-1, cell counting assay, and observation of morphological changes were performed to evaluate the cytotoxic effect of linifanib in SKOV3 cells. We analyzed SKOV3 cells treated with linifanib using Muse cell analyzer. We focused on investigating the effect of linifanib on DNA damage in nucleus. Additionally, intracellular reactive oxygen species (ROS) level was measured through Muse cell analyzer. Western blotting was performed to evaluate the protein expression level related to apoptosis. We found that linifanib inhibited proliferation of SKOV3 cells. Our results showed that linifanib induced apoptosis in SKOV3 cells. Additionally, linifanib induced DNA damage in SKOV3 cells. We found that intracellular ROS level increased after treatment of linifanib in SKOV3 cells. Interestingly, FOXO3 was transferred from cytosol into nucleus after linifanib treatment. Taken together, our results supported that linifanib inhibited the proliferation of human ovary cancer SKOV3 cells, which suggested that linifanib might have the potential to be developed as drugs for ovarian cancer treatment.     

Photogeneration and quenching of reactive oxygen species by urocanic acid

Urocanic acid, UCA, is characterized by two electronic transitions in the UV-B (280-320 nm) which comprise its broad absorption spectrum and give rise to wavelength-dependent isomerization quantum yields. The absorption spectrum of UCA extends into the UV-A (320-400 nm). Given the UV-A component of sunlight is significantly greater than the UV-B component it is hypothesized even weak UV-A photochemistry of UCA could be important for in vivo responses to UV radiation. Degenerate pump-probe experiments performed on t-UCA at several wavelengths in the UV-A reveal an excited-state absorption that undergoes a rapid, approximately 1 ps decay. Photoacoustic experiments performed on both the cis and trans isomers reveal the formation of a long-lived intermediate following UV-A excitation. The efficiency and action spectra for this latter photoactive process are presented and are similar for both isomers of UCA. Cholesterol hydroperoxide assays designed to investigate the nature of the UV-A photoreactivity of t-UCA confirm the production of reactive oxygen species. The bimolecular rate constant for the quenching of singlet oxygen by t-UCA is determined to be 3.5 x 10(6) M(-1) s(-1). Taking into consideration recent theoretical calculations and jet expansion studies of the electronic structure of gas-phase t-UCA, a model is proposed to explain the isomerization and photoreactivity of t-UCA in solution over the UV-A region.     

Nanozymes for regulation of reactive oxygen species and disease therapy

With high catalytic activity and stability, nanozymes have huge advantage in generating or eliminating the reactive oxygen species (ROS) due to their intrinsic enzyme-mimicking abilities, therefore attracting wide attention in ROS-related disease therapy. To better design nanozyme-based platforms for ROS-related biological application, we firstly illustrate the catalytic mechanism of different activities, and then introduce different strategies for using nanozymes to augment or reduce ROS level for the applications in cancer therapy, pathogen infection, neurodegeneration, etc. Finally, the challenges and future opportunities are proposed for the development and application of nanozymes.     

Unmodified cadmium telluride quantum dots induce reactive oxygen species formation leading to multiple organelle damage and cell death

Quantum dots (QDs) are luminescent nanoparticles with unique optical properties that have been exploited for single-cell and whole-animal imaging. When coated with proteins or biocompatible polymers, QDs are not deleterious to cells and organisms. However, when QDs are retained in cells or accumulated in the body for a long period of time, their coatings may be degraded, yielding "naked" QDs. Here, we show that "naked" QDs induce damage to the plasma membrane, mitochondrion, and nucleus, leading to cell death. Reactive oxygen species (ROS) are important players in mediating QD-induced cellular damage. QD-induced cytotoxicity can be reduced or even eliminated without covalent binding of protective agents to the QD surface. Results from these studies suggest the critical role of several subcellular compartments in QD-induced cytotoxicity and point toward multiple molecular targets in nonclassical apoptosis.