ascorbic-acid and 2--7--dichlorofluorescein

Atmospheric processing of carbonaceous nanoparticles (CNPs) may play an important role in determining their fate and environmental impacts. This work investigates the reaction between aerosolized C60 and atmospherically relevant mixing ratios of O3 at differing levels of humidity. Results indicate that C60 is oxidized by O3 and forms a variety of oxygen-containing functional groups on the aerosol surface, including C60O, C60O2, and C60O3. The pseudo-first-order reaction rate between C60 and O3 ranges from 9 × 10(-6) to 2 × 10(-5) s(-1). The reaction is likely to be limited to the aerosol surface. Exposure to O3 increases the oxidative stress exerted by the C60 aerosols as measured by the dichlorofluorescein acellular assay but not by the uric acid, ascorbic acid, glutathione, or dithiothreitol assays. The initial prevalence of C60O and C60O2 as intermediate products is enhanced at higher humidity, as is the surface oxygen content of the aerosols. These results show that C60 can be oxidized when exposed to O3 under ambient conditions, such as those found in environmental, laboratory, and industrial settings.

Topics: Aerosols; Ascorbic Acid; Chemistry Techniques, Analytical; Fluoresceins; Fullerenes; Glutathione; Humidity; Oxidation-Reduction; Oxygen; Ozone

Iron chelators of the 2'-benzoylpyridine thiosemicarbazone (BpT) class show substantial potential as anticancer agents. To explore structure-activity relationships, new BpT analogues were designed that incorporated halogen substituents on the noncoordinating phenyl group (XBpTs). These XBpT ligands exhibited potent antiproliferative activity with some analogues exceeding that of the parent BpT compound. Importantly, there was an appreciable therapeutic index in vitro, as mortal cells were significantly less affected by these chelators relative to neoplastic cells. The addition of a halogen led to a halogen-specific increase in the redox potential of XBpT-Fe complexes. Probing for chelator-induced intracellular reactive oxygen species (ROS) with the fluorescent probe, 2',7'-dichlorofluorescein, revealed a 1.5-4.7-fold increase in fluorescence upon incorporation of Cl, Br, or I to the parent analogues. Furthermore, an important structure-activity relationship was deduced where the addition of halogens led to a positive correlation between intracellular ROS generation and antiproliferative activity in the more hydrophilic BpT parent compounds.

Topics: Antineoplastic Agents; Ascorbic Acid; Cell Line; Cell Line, Tumor; Cell Proliferation; Coordination Complexes; Crystallography, X-Ray; Drug Screening Assays, Antitumor; Electrochemistry; Fluoresceins; Fluorescent Dyes; Fluorometry; Humans; Hydrophobic and Hydrophilic Interactions; Iron Chelating Agents; Molecular Structure; Oxidation-Reduction; Pyridines; Reactive Oxygen Species; Structure-Activity Relationship; Thiosemicarbazones

Reactive oxygen species (ROS) have been implicated in many ionizing radiation-related phenomena, including bystander effects. The oxidation of 2'7'-dichlorofluorescin (DCFH) to fluorescent 2'7'-dichlorofluorescein (DCF) is commonly used for the detection of radiation-induced ROS. The DCF assay was adapted for efficient, systematic flow cytometry quantification of low-linear energy transfer (LET) gamma-radiation-induced ROS in vitro in Chinese hamster ovary (CHO) cells. This method is optimized for increased sensitivity to radiation-induced ROS and to discriminate against measurement of extracellular ROS. This method can detect a significant increase in ROS in cells exposed to gamma radiation at doses as low as 10 cGy. The antioxidants N-acetyl-cysteine and ascorbic acid (vitamin C) significantly reduced the amount of ROS measured in cells exposed to 5 Gy ionizing radiation. This method was used to measure the intracellular ROS in unirradiated CHO bystander cells co-cultured with low-LET-irradiated cells. No increase in ROS was measured in bystander cell populations co-cultured with the irradiated cells beginning 9 s after radiation exposure.

Topics: Acetylcysteine; Animals; Ascorbic Acid; Bystander Effect; CHO Cells; Cricetinae; Cricetulus; Dose-Response Relationship, Radiation; Flow Cytometry; Fluoresceins; Reactive Oxygen Species

To evaluate the protective effects of antioxidant agents against space radiation-induced oxidative stress in cultured human epithelial cells.. The effects of selected concentrations of N-acetylcysteine, ascorbic acid, sodium ascorbate, co-enzyme Q10, alpha-lipoic acid, l-selenomethionine, and vitamin E succinate on radiation-induced oxidative stress were evaluated in MCF10 human breast epithelial cells exposed to radiation with X-rays, gamma-rays, protons, or high mass, high atomic number, and high energy particles using a dichlorofluorescein assay.. The results demonstrated that these antioxidants are effective in protecting against radiation-induced oxidative stress and complete or nearly complete protection was achieved by treating the cells with a combination of these agents before and during the radiation exposure.. The combination of antioxidants evaluated in this study is likely be a promising countermeasure for protection against space radiation-induced adverse biologic effects.

Topics: Acetylcysteine; Antioxidants; Ascorbic Acid; Cells, Cultured; Coenzymes; Epithelial Cells; Fluoresceins; Humans; Oxidative Stress; Radiation Protection; Selenomethionine; Thioctic Acid; Tocopherols; Ubiquinone; Vitamin E

To examine the effect of oxidized low-density lipoprotein (ox-LDL) on the intracellular production of reactive oxygen species (ROS) in bovine articular chondrocytes (BACs) and to investigate whether this increase occurs through binding to the receptor lectin-like ox-LDL receptor-1 (LOX-1). Furthermore, to ascertain whether the binding of ox-LDL to LOX-1 results in NF-kappaB activation.. BACs were preincubated with 2',7'-dichlorofluorescin diacetate (DCFH-DA), a dye that allows the monitoring of intracellular ROS production for DCF by spectrofluorometry. BACs were incubated with native LDL and ox-LDL (10, 50, and 100 microg/ml) for 5 min at 37 degrees C and DCF formation was observed. BACs were also preincubated with anti-LOX-1 mAb (40 microg/ml) or ascorbic acid (10 microM). Nuclear extracts from BACs treated for the indicated periods with 50 microg/ml ox-LDL, and preincubated with anti-LOX-1 mAb or ascorbic acid, were prepared and analyzed by electrophoretic mobility shift assay (EMSA).. ox-LDL induced a significant dose-dependent increase in ROS production after 5-min incubation with BACs (P < 0.001). ROS formation was markedly reduced in BACs preincubated with anti-LOX-1 mAb and ascorbic acid (P < 0.001). Activation in BACs of the transcription factor NF-kappaB was evident after 5-min incubation with ox-LDL and was attenuated by anti-LOX-1 mAb and ascorbic acid.. ox-LDL binding to LOX-1 in BACs increased the production of intracellular ROS and activated NF-kappaB. Reduction of NF-kappaB activation by ascorbic acid indicates that the activation, at least in part, is ROS-dependent. These observations support the hypothesis that hypercholesterolemia is one of several risk factors for arthritis, and that lipid peroxidation products such as ox-LDL are involved in cartilage matrix degradation.

Topics: Animals; Ascorbic Acid; Cartilage, Articular; Cattle; Cells, Cultured; Chondrocytes; Dose-Response Relationship, Drug; Fluoresceins; Lipoproteins, LDL; Microscopy, Confocal; NF-kappa B; Oxidation-Reduction; Reactive Oxygen Species; Receptors, LDL; Receptors, Oxidized LDL; Reverse Transcriptase Polymerase Chain Reaction

Hypericin is a photosensitizing pigment found in St. John's wort (Hypericum perforatum) displaying a high toxicity towards certain tumors. The fact that some non-tumor cells, especially monocytes and granulocytes, are resistant to its photocytotoxic effects, posed the question whether this insensitivity is due to their ability to accumulate vitamin C, an antioxidant which alleviates the deleterious work of free radicals. HL-60 promyelocytic tumor cells can be differentiated to neutrophilic granulocytes by treatment with dimethylsulfoxide and were used as cell model. In the differentiated cells, treatment with phorbol esters (PMA) stimulates vitamin C (ascorbate) transport. The uptake rates were unaltered by hypericin at concentrations below 1 microM and irradiation with visible light at a light dose of 6 J/cm2. Inhibition by higher concentrations of hypericin was most probably due to a combination of photocytotoxic properties of the dye and oxygen radicals generated during respiratory burst. Superoxide production by NADPH oxidase followed by reduction of ferricytochrome c was inhibited by hypericin. The degree of inhibition was dependent on the concentration of hypericin and light intensity: IC50-values were 1.7 and 0.7 microM under light doses of 3.6 and 10.8 J/cm2, respectively. Oxidative stress, monitored with 2',7'-dichlorofluorescein (DCF) was only slightly decreased by ascorbate even at higher concentrations of hypericin. In contrast to its effect on the ferricytochrome c-reduction, irradiation had no significant influence on DCF-fluorescence. However, the viability of the cells was strongly decreased after photosensitization and no significant improvement was obtained by ascorbate. Results from this work indicate that ascorbate transport per se is not altered during photodynamic therapy and vitamin C does not interfere with hypericin-induced photodamage of cellular targets.

Topics: Anthracenes; Antineoplastic Agents; Antioxidants; Ascorbic Acid; Biological Transport; Cell Differentiation; Cytochromes c; Fluoresceins; HL-60 Cells; Humans; Light; NADPH Oxidases; Oxidation-Reduction; Oxidative Stress; Perylene; Phorbol Esters; Photochemotherapy; Protein Kinase C; Reactive Oxygen Species; Respiratory Burst; Superoxides

The release of cytochrome c from mitochondria is a crucial step in apoptosis, resulting in the activation of the caspase proteases. A further consequence of cytochrome c release is the enhanced mitochondrial production of superoxide radicals (O2.), which are converted to hydrogen peroxide by manganese-superoxide dismutase. Recently, we showed that cytochrome c is a potent catalyst of 2',7'-dichlorofluorescin oxidation to the fluorescent 2',7'-dichlorofluorescein by these species, leading to the conclusion that 2',7'-dichlorofluorescein fluorescence is a reflection of cytosolic cytochrome c concentration rather than "reactive oxygen species" levels (Burkitt, M. J., and Wardman, P. (2001) Biochem. Biophys. Res. Commun. 282, 329-333). The oxidant generated from cytochrome c has so far not been identified. Several authors have suggested that the hydroxyl radical (*OH) is generated, but others have discussed the possibility of a peroxidase compound I. By examining the effects of various antioxidants (glutathione, ascorbate, and NADH) and "hydroxyl radical scavengers" (ethanol and mannitol) on the rate of 2',7'-dichlorofluorescin oxidation by cytochrome c, together with complementary EPR spin-trapping studies, we demonstrate that the hydroxyl radical is not generated. Instead, our findings suggest the formation of a peroxidase compound I-type intermediate, in which one oxidizing equivalent is present as an oxoferryl heme species and the other as the protein tyrosyl radical previously identified (Barr, D. P., Gunther, M. R., Deterding, L. J., Tomer, K. B., and Mason, R. P. (1996) J. Biol. Chem. 271, 15498-15503). Competition studies involving spin traps indicated that the oxoferryl heme component is the active oxidant. These findings provide an improved understanding of the physicochemical basis of the redox changes that occur during apoptosis.

Topics: Animals; Apoptosis; Ascorbic Acid; Cell Death; Cytochrome c Group; Electron Spin Resonance Spectroscopy; Ethanol; Fluoresceins; Free Radicals; Glutathione; Heme; Horses; Hydrogen Peroxide; Models, Chemical; NAD; Oxidation-Reduction; Oxidative Stress; Oxygen; Reactive Oxygen Species; Temperature; Time Factors

beta-Amyloid protein is the major component of senile plaques found in the brains of Alzheimer's patients. Previously, a new biochemical property of amyloid, its ability to disrupt ester and peptide bonds, was described [Elbaum, D., Brzyska, M., Bacia, A. & Alkon, D. (2000) Biochem. Biophys. Res. Commun. 267, 733-738]. In the present work we compare the ability of beta-amyloid to hydrolyse and oxidize model fluorescent derivatives of dichlorofluorescein [dichlorodihydrofluorescein (H2DCF) or dichlorofluorescein diacetate (DCF-DA), respectively] to the same final product (dichlorofluorescein). Although there is accumulating evidence of oxidative properties of beta-amyloid, little is known about its hydrolytic abilities. Chemical modification studies revealed that hydrolytic properties are related to a His, Ser and Asp/Glu triad, while residues of His, Tyr and Met are involved in the oxidative activity of amyloid. Studies with the rat homologue of human beta-amyloid (1-40), containing three amino-acid substitutions (Arg5-->Gly, Tyr10-->Phe and His13-->Arg) confirmed a role of His in the studied processes. Reduction of the hydrolysis product caused by inhibitors of Ser esterases (phenylmethylsulphonyl fluoride and eserine) suggests that beta-amyloid-mediated hydrolysis is Ser sensitive. Antioxidants and metal chelators that reduced H2DCF oxidation did not change or increase DCF-DA hydrolysis. Solvent isotope effects suggest the involvement of hydrogen bonds in the hydrolysis reaction. Hydrolysis was inhibited by redox-active metal ions and was practically oxygen independent while the oxidation process was redox-active-metal enhanced [Cu(II) and Fe(II) primarily], and oxygen dependent. Product formation was significantly inhibited by catalase and superoxide dismutase as well as benzoquinone, a specific superoxide anion radical scavenger. Increase of fluorescence by oxidation was strongly inhibited by azide and His and enhanced in samples prepared with deuterated phosphate buffer, suggesting singlet oxygen intermediacy. Our data are consistent with superoxide-mediated singlet oxygen intermediate in this Fenton mechanism-driven reaction. These results indicate that hydrolytic and oxidative properties of beta-amyloid are distinct features of this peptide and probably require different mechanisms to occur, but both of them may contribute to beta-amyloid toxicity.

Topics: Amino Acid Sequence; Amyloid beta-Peptides; Animals; Ascorbic Acid; Dose-Response Relationship, Drug; Fluoresceins; Fluorescent Dyes; Humans; Hydrolysis; Molecular Sequence Data; Oxidation-Reduction; Rats; Vitamin E

After physically disrupting cell contacts, apoptosis of bursal cells of Fabricius was induced during in vitro cultivation. The percentage of apoptotic cells increased with incubation time and approximately 70% cells represented apoptosis after 6 hr of incubation. The induction of apoptosis was significantly inhibited by treatment of the cells with ascorbic acid (vitamin C), but not with trolox, a vitamin E analog. An intense DNA ladder pattern was shown at 6 hr post-isolation, which is a biochemical hallmark of apoptosis. Treatment of the cells with ascorbic acid inhibited the DNA fragmentation, but trolox did not. To monitor the intracellular production of reactive oxygen species (ROSs), the intensity of fluorescence emitted from DCFH-DA was measured. The intensity of fluorescence from cells incubated for 0.5-2 hr was approximately 2-fold higher than that from cells at 0 hr. The relative intensity of fluorescence decreased immediately after the addition of ascorbic acid to the cells. The intensity from the cells treated with ascorbic acid was 20-30% of that from the control cells at each incubation time. For trolox, the intensity was 50-70% of that from the control cells at each 1 to 2 hr incubation time. When ROSs-induced lipid peroxidation was assessed using cis-parinaric acid (PnA) as a monitor molecule, lipid peroxidation was found to occur in the control cells after isolation of the bursal cells. Treatment of the cells with trolox reduced lipid peroxidation, but treatment with ascorbic acid enhanced peroxidation.

Topics: Animals; Antioxidants; Apoptosis; Ascorbic Acid; Bursa of Fabricius; Cells, Cultured; Chickens; Chromans; DNA Fragmentation; Electrophoresis, Agar Gel; Fatty Acids, Unsaturated; Fluoresceins; Fluorescent Dyes; Lipid Peroxidation; Male; Reactive Oxygen Species; Spectrometry, Fluorescence

The inhibitory effects of N-(4-hydroxyphenyl)retinamide (4HPR) on the process of carcinogenesis are not fully understood and may result from its ability to induce apoptosis in transformed cells. This study investigated the apoptotic properties of 4HPR in four human cutaneous squamous cell carcinoma cell lines. Apoptosis induction, detected by the terminal deoxynucleotidyl transferase dUTP nick end labeling method, occurred in a dose- and time-dependent fashion after treatment with 4HPR. 4HPR promoted reactive oxygen species (ROS) determined by oxidation of 2',7'-dichlorofluorescin. 4HPR-induced ROS, and apoptosis could be inhibited by L-ascorbic acid. Rhodamine 123 retention revealed that 4HPR treatment promoted a gradual dissipation of mitochondrial inner transmembrane potential, and this could be inhibited by L-ascorbic acid, implying that mitochondrial permeability transition was involved in apoptosis induction. Cyclosporin A and bongkrekic acid inhibited dissipation of mitochondrial inner transmembrane potential, ROS production, and DNA fragmentation after exposure to 4HPR, demonstrating that mitochondrial permeability transition was a central coordinating feature of 4HPR-induced apoptosis.

Topics: Anticarcinogenic Agents; Apoptosis; Ascorbic Acid; Carcinoma, Squamous Cell; Cell Division; Cyclosporine; DNA Fragmentation; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; Enzyme Inhibitors; Fenretinide; Fluoresceins; Fluorescent Dyes; Humans; Intracellular Membranes; Membrane Potentials; Mitochondria; Oxidation-Reduction; Permeability; Reactive Oxygen Species; Rhodamine 123; Skin Neoplasms; Tumor Cells, Cultured

Using reaggregating rat brain cell cultures at two different stages of differentiation, we examined the biochemical effects of a 10-day treatment with nanomolar concentrations of methylmercuric chloride (monomethylmercury), in the presence or absence of promoters of hydroxyl radical formation (10 microM copper sulphate plus 100 microM ascorbate). A decrease in total protein content accounted for the general cytotoxicity of these compounds, whereas selective effects were assessed by determining the activities of cell type-specific enzymes. Methylmercury, up to 100 nM, as well as the copper ascorbate mixture, when applied separately, induced no general cytotoxicity, and only slight effects on neuronal parameters. However, when applying 100 nM methylmercury and the copper-ascorbate mixture together, a drastic decrease in neuronal and glial parameters was found. Under these conditions, the content of reactive oxygen species, assessed by 2',7'-dichlorofluorescin oxidation, increased greatly, while the activities of antioxidant enzymes decreased. In the presence of copper and ascorbate, differentiated cultures appeared more resistant than immature ones to low methylmercury concentrations (1-10 mM), but did undergo similar changes in both cell type-specific and antioxidant enzyme activities at 100 nM methylmercury. These results suggest that in prooxidant conditions low doses of mercury can become much more deleterious for the central nervous system.

Topics: Animals; Ascorbic Acid; Brain; Cells, Cultured; Copper Sulfate; Drug Combinations; Drug Resistance; Fluoresceins; Hydroxyl Radical; Methylmercury Compounds; Neuroglia; Neurons; Osmolar Concentration; Rats; Reactive Oxygen Species

Exposure of intact cultured Chinese hamster ovary (CHO) cells to water-soluble nickel (Ni) salts and to relatively water-insoluble crystalline nickel subsulfide (Ni3S2) resulted in an increased formation of the fluorescent oxidized compound, dichlorofluorescein (DCF) from the parent nonfluorescent compound, 2,7-dichlorofluorescin diacetate. This fluorescent product was also formed in vitro following oxidation with relatively strong oxidants such as hydrogen peroxide in the presence of peroxidase, suggesting that Ni increased the concentration of hydrogen peroxide in intact cells. However, formation of other strong oxidants such as hydroperoxides is possible since they have also been shown to cause the oxidation of the nonfluorescent dichlorofluorescin to the fluorescent product DCF in vitro. Localization of the oxidized fluorescent DCF in intact cells was also examined by fluorescence microscopy. Both Ni3S2 and NiCl2 appeared to increase the degree of fluorescence in intact CHO cells around the nuclear membranes. This increase in fluorescence was greater in the presence of relatively water-insoluble Ni3S2 than water-soluble NiCl2. These results add to the emerging concept that Ni-induced genotoxicity may be mediated by oxygen radical intermediates.

Topics: Amitrole; Animals; Ascorbic Acid; Cell Survival; CHO Cells; Cricetinae; Fluoresceins; Fluorescence; Horseradish Peroxidase; Nickel; Oxidants