Rapid upregulation of cytoprotective nitric oxide in breast tumor cells subjected to a photodynamic therapy-like oxidative challenge

Many tumor cells produce nitric oxide (NO) as an antiapoptotic/progrowth molecule which also promotes antiogenesis and tumor expansion. This study was designed to examine possible antagonistic effects of endogenous NO on tumor eradication by photodynamic therapy (PDT). Using COH-BR1 breast cancer cells sensitized in mitochondria with 5-aminolevulinic acid (ALA)-generated protoporphyrin IX as a model for ALA-based PDT, we found that caspase-9 activation and apoptotic death following irradiation were strongly enhanced by 1400W, an inhibitor of inducible nitric oxide synthase (iNOS). RT-PCR and Western analyses revealed a substantial upregulation of both iNOS mRNA and protein, beginning ca 4 h after irradiation and persisting for at least 20 h. Accompanying this was a strong 1400W-inhibitable increase in intracellular NO, as detected with the NO probe, DAF-2-DA. Short hairpin RNA-based iNOS knockdown in COH-BR1 cells dramatically reduced NO production under photostress while enhancing caspase-9 activation and apoptosis. These findings suggest that cytoprotective iNOS/NO induction in PDT-treated tumor cells could reduce treatment efficacy, and point to pharmacologic intervention with iNOS inhibitors for counteracting this.     

Chain-breaking antioxidant activity of phosphite esters

The inhibition of the autoxidation of hydrocarbons and polypropylene by aliphatic, aromatic, sterically hindered and cyclic phosphites has been studied by means of volumetric and ³¹P-NMR techniques. The antioxidant activity of phosphites depends on the rate of their reactions with peroxyl radicals and on the way they react with alkoxyl radicals. Only those phosphites which react by substitution to give free aryloxyl radicals are effective as chain-breaking antioxidants.The reaction modes of various phosphites with various peroxyl and alkoxyl radicals have been studied in some model reactions and the relationship between structure, reaction mechanism and antioxidant activity has been elucidated.     

Electron transfer mechanism for regulatory action by nitric oxide

Various mechanisms have been proposed for the regulatory action of nitric oxide. The most common mechanism involves stimulation of soluble guanylate cyclase (sGC) with consequent elevation of cyclic GMP levels. A more detailed chemical mode of action is given in this paper entailing electron transfer (ET) processes which ensue after binding of NO to iron-containing enzymes, and which may be responsible for some of the observed physiological effects. The focus is on immunological reactions and neurotransmission. Electrochemical data for model FeNO complexes are discussed which support the hypothesis. Comparisons are made with the mode of action by anti-infective drugs. The ET transformations suggested for NO in the nervous system are in marked contrast with the current concentration on ionic reactions. Based on the ET framework, analogies exist with a proposed mechanism for the plant hormone ethylene. Other supportive evidence from various categories is also presented. The general approach in this paper represents an elaboration of the one-electron redox processes put forth recently for NO action by Saran and Bors (Chem.-Biol. Interact., 90 (1994) 35).     

Detection of nitric oxide in single cells

Nitric oxide (NO) is endogenously generated by nitric oxide synthase (NOS) enzymes and is involved in a surprisingly wide range of biological functions. As efforts are made to elucidate the regulatory mechanisms of NOS expression and function, there is increasing interest in following NOS activity directly by monitoring NO production. Additionally, spatial and temporal measurements of NO are important for understanding its function and metabolism. In this work, developments in technology enabling NO detection in biological systems are reviewed. Measuring NO at single cell levels is important as NOS is heterogeneously distributed; however, such measurements are difficult as physiological NO levels are in the low nanomolar to low micromolar range. Here, three categories of analytical techniques enabling NO detection at single cell levels are highlighted: fluorescence microscopy, capillary electrophoresis with laser induced fluorescence detection, and electrochemistry. For each, the basic principles, performance, applications, figures of merits and limitations are presented in terms of single cell NO detection.     

Generation, translocation, and action of nitric oxide in living systems

Nitric oxide (NO) is a gaseous diatomic radical that is involved in a wide range of physiological and pathological functions in biology. Conceptually, the biochemistry of NO can be separated into three stages: generation (stage 1), translocation (stage 2), and action (stage 3). In stage 1 the oxygenase domain of NO synthase converts L-arginine to L-citrulline and NO (g). Owing to its short-lived nature, this molecule is converted into a different nitrogen oxide such as NO(2), an organonitrosyl such as a nitrosothiol, or a metal nitrosyl such as a heme-nitrosyl, for transportation in stage 2. Each of these derivatives features unique physical characteristics, chemical reactivity, and biological activity. Upon delivery in stage 3, NO exerts its physiological or pathological function by reaction with biomolecules containing redox-active metals or other residues.     

Activation of macrophage tumoricidal activity by photodynamic treatment in vitro-indirect activation of macrophages by photodynamically killed tumor cells

Macrophages constitute a major part of natural tumor defense by their capacity to destroy selectively a broad range of tumor types upon specific activation. In the last couple of years, these cells have also been implicated as effector cells in the destruction of tumors by photodynamic therapy. In the present work, the potential role of macrophage-mediated tumor cytotoxicity after photodynamic treatment in vitro has been investigated with respect to photodynamic activation of macrophages for tumoricidal effector functions. Our data show that photodynamic treatment of highly pure murine bone-marrow-derived macrophages with the hematoporphyrin derivative Photosan-3 does not result in activation of these cells for cytotoxicity against YAC-1 tumor cells or secretion of tumor necrosis factor and nitric oxide, irrespective of co-stimulation with interferon-γ, a potent priming agent for macrophage antitumoral activity. On the contrary, treatment with higher photosensitizer doses is found to reduce markedly the viability of the macrophage effector cells. Thus, these results do not lend any support to the hypothesis of direct macrophage activation by photodynamic treatment. However, macrophages are found to be activated for tumoricidal effector functions indirectly by photodynamically killed tumor cells, in a way reminiscent of phagocytosis-inducing stimuli. It is thus suggested that recognition and phagocytosis of photodynamically destroyed tumor cells constitutes the major signal for local activation of macrophages in photodynamically treated tumor tissues, which may be crucial for final, specific eradication by the immune system of tumor cells surviving photodynamic treatment.     

Protective effect of mitochondrially targeted antioxidant MitoQ on oxidatively stressed fibroblasts

3T3 and VH10 cells were subjected to oxidative damage by hydroxyl radicals generated from Cu(II) ions and ascorbate. The presence of reactive oxygen and nitrogen species in cells was determined using fluorescent dyes. MitoQ—a mitochondrially targeted antioxidant—was examined to prevent cell death and was compared with trolox as a reference standard serving as an antioxidant. MitoQ at lower concentrations (up to 500 nM) prevented damage of mitochondria and thereby increased viability of cells. However, at higher concentrations (over 500 nM) MitoQ decreased the viability of both strains of cells. The last observation indicates that the application of MitoQ should be evaluated also from the point of its cell-destructive potential.     

Protein thiyl free radicals produced by nitric oxide and nitric oxide donors

The spin-trapping technique was used to study the radical intermediates produced by reaction of nitric oxide (^*NO) and peroxynitrite with serum albumin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Our results show that the major radical product induced by ^*NO and by peroxynitrite with serum albumin and GAPDH was a thiyl radical. The same radical can be detected in the ^*NO-transfer from S-nitroso albumin to low molecular weight thiols. Moreover, ^*NO or peroxynitrite treatment of GAPDH was able to induce NAD-dependent covalent modification of the enzyme in erythrocyte ghosts.     

Seminaphthofluorescein-based fluorescent probes for imaging nitric oxide in live cells

Fluorescent turn-on probes for nitric oxide based on seminaphthofluorescein scaffolds were prepared and spectroscopically characterized. The Cu(II) complexes of these fluorescent probes react with NO under anaerobic conditions to yield a 20-45-fold increase in integrated emission. The seminaphthofluorescein-based probes emit at longer wavelengths than the parent FL1 and FL2 fluorescein-based generations of NO probes, maintaining emission maxima between 550 and 625 nm. The emission profiles depend on the excitation wavelength; maximum fluorescence turn-on is achieved at excitations between 535 and 575 nm. The probes are highly selective for NO over other biologically relevant reactive nitrogen and oxygen species including NO(3)(-), NO(2)(-), HNO, ONOO(-), NO(2), OCl(-), and H(2)O(2). The seminaphthofluorescein-based probes can be used to visualize endogenously produced NO in live cells, as demonstrated using Raw 264.7 macrophages.     

l-Arginine and nitric oxide synthesis in the cells with inducible NO synthase

Russian Chemical Bulletin - The effect of citrulline and ammonium chloride on the nitric oxide formation by peritoneal macrophages and liver tissue cells was studied using ESR spectroscopy. In ex...     

Nitrogen isotope exchange between nitric oxide and nitric acid

The rate of nitrogen isotope exchange between NO and HNO₃ has been measured as a function of nitric acid concentration of 1.5–4M·1^–1. The exchange rate law is shown to beR=k[HNO₃]²[N₂O₃] and the measured activation energy isE=67.78kJ ·M^–1 (16.2 kcal·M^–1). It is concluded that N₂O₃ participates in¹⁵N/¹⁴N exchange between NO and HNO₃ at nitric acid concentrations higher than 1.5M·1^–1.     

Discovery of nitric oxide in marine ecological system and the chemical characteristics of nitric oxide

Nitric oxide was discovered in both the lab and the alga culture pond of Daya Bay (1―300 m3) before the growth of alga reached the maximum. The results included: (1) NO was detected before the growth of alga reached the maximum in the case of red tide alga and food alga, and the concentration of NO decreased rapidly after the growth maximum; (2) the curve between NO con-centration and time indicated that the concentration of NO in the daytime was more than that at night, and the maximal concentration of NO appeared in the midday (1―3 pm); (3) the growth of alga reached the maximum in the alga culture pond of Daya Bay in about 8―10 d, and NO was discovered in 5―7 d; (4) the measured NO concentration was 10-9 mol/L, 10-9―10-8 mol/L, and 10-8 mol/L for Haeterosigma akashiwo, mixed alga in Daya Bay and Chaetoceros Curvisetus individually; (5) the relation of illumination with NO production was discussed.     

Effectiveness of nitric oxide ozonation

Attempts to develop new technologies of NO _x (NO + NO₂) emission reduction are still carried out all around the world. One of the relatively new approaches is the application of ozone injection into the exhaust gas stream followed by the absorption process. Ozone is used to transform NO _x to higher nitrogen oxides which yield nitric acid with better effectiveness. The main objective of this paper was to study the influence of mole ratio (MR) O₃/NO used in the ozonation process of NO _x on the effectiveness of NO _x oxidation to higher oxides. The ozonation process was carried out in a flow reactor for concentrations of nitric oxide in the range of 1.5 × 10⁻⁵−7.7 × 10⁻⁵ mol dm⁻³ and varying O₃/NO mole ratios. Measurements were conducted with the use of a FTIR spectrometer. The results obtained prove that for MR higher than 1, the oxidation effectiveness of nitric oxides generally reaches 95 %, whereas for MR higher than 2, oxidation of NO _x to higher nitrogen oxides is completed.     

The contribution of fructose and nitric oxide to oxidative stress in hamster islet tumor (HIT) cells through the inactivation of glutathione peroxidase

Reducing sugars, such as glucose and fructose, are known to play a role in the initiation of apoptosis in pancreatic beta-cells. The data collected in this study show that fructose increases H2O2 levels and lipid peroxidation of hamster islet tumor (HIT) cells, which originated from hamster pancreatic beta-cells. In an attempt to clarify the mechanism of this oxidative stress, we were able to show that glutathione peroxidase (GPx) is inactivated by fructose, and that mRNA expression of GPx is suppressed by fructose. Nitric oxide (NO) is also known to bring about apoptosis. The presence of NO increases intracellular peroxide levels in HIT cells as judged by flow cytometric analysis. These data suggest that fructose and nitric oxide suppress the activity or expression of GPx, and, as a result, permit an increase in intracellular peroxides or lipid peroxidation. This represents a major contribution to the main mechanism of apoptosis by fructose and NO.     

Effect of temperature on biofiltration of nitric oxide

Applied Biochemistry and Biotechnology -     

Kinetics of nitric oxide oxidation

Nitrogen oxides are nowadays a subject of global concern. Several types of nitrogen oxides exist in the environment: N₂O, NO, NO₂, N₂O₃, N₂O₄, N₂O₅. The abbreviation NO _x usually relates to nitric oxide NO, nitrogen dioxide NO₂, and nitrous oxide N₂O. The first two are harmful pollutants for both environment and human health, whereas the third is one of the greenhouse gases. Implementation of stringent NO _x emission regulations requires the development of new NO _x removal technologies from exhaust gases. One of many proposals for NO _x emission reduction is the application of an oxidizing agent which would transform NO _x to higher nitrogen oxides with higher solubility in water. The main objective of the paper was to present the rate constant of nitric oxide oxidation, determined in our studies.     

Biomimetic and microbial reduction of nitric oxide

The biomimetic reduction of nitric oxide (NO) to nitrous oxide (N₂O) by dithiothreitol in the presence of cyanocobalamin and cobaltcentered porphyrins has been investigated. Reactions were monitored directly using Fourier Transform Infrared (FTIR) Spectroscopy vapor-phase spectra. Reaction rates were twofold faster for the corrin than for the cobalt-centered porphyrins. The stoichiometry showed the loss of two molecules of NO per molecule of N₂O produced. We have also demonstrated that the facultative anaerobe and chemoautotroph,Thiobacillus denitrificans, can be cultured anoxically in batch reactors using NO as a terminal electron acceptor with reduction to elemental nitrogen (N₂). We have proposed that the concentrated stream of NO_x, as obtained from certain regenerable processes for the gas desulfurization and NO_x removal, could be converted to N₂ for disposal by contact with a culture ofT. denitrificans. Four heterotrophic bacteria have also been identified that may be grown in batch cultures with succinate, yeast extract, or heat and alkali pretreated sewage sludge as carbon and energy sources and NO as a terminal electron acceptor. These areParacoccus dentrificans, Pseudomonas denitrificans, Alcaligens denitrificans, andThiophaera pantotropha.     

Novel nitric oxide microsensor and its application to the study of smooth muscle cells

A novel sensitive, selective and stable nitric oxide (NO) microsensor is described, which is modified by nano Au colloid and Nafion. As determined by atomic forced microscopy (AFM), the diameter of Au colloid particles is from 7 to 14 nm. The detection of NO is based on the nano Au particles catalysis of NO oxidation at an anodic potential of +0.74 V (versus saturated calomel electrode (SCE)). The microsensor showed a low detection limit, high selectivity and sensitivity for NO determination. The oxidation current (measured by differential pulse amperometric technique) was linear with NO concentration ranging from 1.0×10⁻⁷ to 4.0×10⁻⁵ mol/l with a calculated detection limit of 5.0×10⁻⁸ mol/l (S/N=3). Using the microsensor, the direct real time production of NO in the smooth muscle cells was continuously measured, which showed the NO levels was increased by stimulating with l-arginine (l-Arg), acetylcholine (Ach) and a self-made flavonoid medicine.     

Highly selective two-photon fluorescent probe for imaging of nitric oxide in living cells简

A new two-photon fluorescent probe, ADNO, for nitric oxide （NO） based on intramolecular photoinduced electron transfer （PET） mechanism d/splays a rapid response to NO with a remarkable fluorescent enhancement in PBS buffer. The excellent chemoselectivity of ADNO for NO over other ROS/RNS （reactive oxygen species or nitrogen species） and common metal ions was observed. Moreover, ADNO has been successfully applied in fluorescence imaging of NO of living cells using both one-photon microscopy （OPM） and two-~hoton microscopy （TPM）,     

Relation between K+ leakage and damage to band 3 in photodynamically treated red cells

Potassium leakage is one of the first events that appear after photosensitization of red blood cells. This event may subsequently lead to colloid osmotic hemolysis. The aim of our study was to determine which photodynamically induced damage is responsible for increased membrane cation permeability. This was done by studying the effect of dimethylmethylene blue (DMMB)-mediated photodynamic treatment (PDT) on different membrane transport systems. Inhibition of band 3 activity (anion transport) showed a comparable light dose dependency as PDT-induced potassium leakage, whereas glycerol transport activity was inhibited only at higher light doses. Dipyridamole (DIP), an inhibitor of anion transport, protects band 3 against DMMB-induced damage, and prevents the increase in cation permeability of the membrane. Damage to glycerol transport was partially reduced when PDT was performed in the presence of DIP. Because DIP has no affinity for the glycerol transporter, this protection might result from the reduced photodamage to band 3. These results support the hypothesis that band 3 might be involved in glycerol transport. Glucose transport was not affected by DMMB-mediated PDT. The present results are the first to show a causal relationship between DMMB-mediated photodamage to band 3 and increased cation permeability of red blood cells.