ascorbic-acid and xylenol-orange

Understanding the mode of action for lipoxygenase (LOX) inhibitors is critical to determining their efficacy in the cell. The pseudoperoxidase assay is an important tool for establishing if a LOX inhibitor is reductive in nature, however, there have been difficulties identifying the proper conditions for each of the many human LOX isozymes. In the current paper, both the 234 nM decomposition (UV) and iron-xylenol orange (XO) assays are shown to be effective methods of detecting pseudoperoxidase activity for 5-LOX, 12-LOX, 15-LOX-1 and 15-LOX-2, but only if 13-(S)-HPODE is used as the hydroperoxide substrate. The AA products, 12-(S)-HPETE and 15-(S)-HPETE, are not consistent hydroperoxide substrates since they undergo a competing transformation to the di-HETE products. Utilizing the above conditions, the selective 12-LOX and 15-LOX-1 inhibitors, probes for diabetes, stroke and asthma, are characterized for their inhibitory nature. Interestingly, ascorbic acid also supports the pseudoperoxidase assay, suggesting that it may have a role in maintaining the inactive ferrous form of LOX in the cell. In addition, it is observed that nordihydroguaiaretic acid (NDGA), a known reductive LOX inhibitor, appears to generate radical species during the pseudoperoxidase assay, which are potent inhibitors against the human LOX isozymes, producing a negative pseudoperoxidase result. Therefore, inhibitors that do not support the pseudoperoxidase assay with the human LOX isozymes, should also be investigated for rapid inactivation, to clarify the negative pseudoperoxidase result.

Topics: Enzyme Assays; Humans; Hydrogen Peroxide; Linoleic Acids; Lipid Peroxides; Lipoxygenase Inhibitors; Lipoxygenases; Oxidation-Reduction; Peroxidase; Phenols; Sulfoxides

Ascorbic acid is an essential micronutrient and is considered to have an antioxidant function in living systems. For the past several decades, ascorbic acid has been the subject of considerable interest as an anticancer agent. Several studies have shown that ascorbic acid is cytotoxic to a variety of cancer cells, whereas normal cells are relatively resistant to such cytotoxic action. In this study, we propose a putative molecular mechanism that accounts for the preferential cytotoxicity of ascorbic acid against cancer cells.. Standard and lysed version of alkaline single-cell gel electrophoresis (Comet assay); ferrous oxidation-xylenol orange (FOX) assay.. We show that ascorbic acid acts as a prooxidant and leads to oxidative DNA breakage in lymphocytes and lymphocyte nuclei. Scavengers of reactive oxygen species were able to inhibit ascorbic acid-induced DNA breakage, suggesting the involvement of reactive oxygen species in this reaction. We further show that such DNA breakage is inhibited by both iron and copper chelators in cells, whereas in nuclei, similar inhibition was achieved only by copper chelators, indicating an important role of chromatin-bound copper in the prooxidant cellular DNA breakage by ascorbic acid.. We propose that the copper-dependent cellular redox status is an important element in the cytotoxic action of ascorbic acid against cancer cells. It is well established that cellular copper levels are considerably elevated in various malignancies. Therefore, cancer cells may be more subject to electron transfer between copper and ascorbate to generate reactive oxygen species. In light of these observations and those in literature, in this paper we explain that the preferential cytotoxicity of ascorbic acid against cancer cells is the result of elevated copper levels in such cells. Further, this study identifies nuclear copper as a novel molecular target for cytotoxic action of ascorbic acid, which has implications for its chemotherapeutic properties against cancer.

Topics: Antioxidants; Ascorbic Acid; Cell Nucleus; Comet Assay; Copper; DNA Breaks; Drug Delivery Systems; Humans; In Vitro Techniques; Lymphocytes; Oxidation-Reduction; Phenols; Reactive Oxygen Species; Sulfoxides; Xylenes

Leaf metabolism produces H2O2 at high rates, but current concepts suggest that the potent signalling effects of this oxidant require that concentrations be controlled by a battery of antioxidative enzymes. The extent to which H2O2 is allowed to accumulate remains unclear. There is little consensus on leaf H2O2 values in the literature and measured concentrations in unstressed conditions range from 50-5000 nmol g(-1) fresh weight, a difference that probably reflects technical inaccuracies as much as biological variability. This article uses new experimental and literature data to examine some of the difficulties in accurately measuring H2O2 in leaf extracts. Potential problems relate to sensitivity, interference from other redox-active compounds, and H2O2 stability during sample preparation. Particular attention is drawn to the influence of tissue mass/extraction volume in the quantitative estimation of H2O2 contents, and the possibility that this factor could contribute to the variability of literature data.

Topics: Adaptation, Physiological; Ascorbic Acid; Chloroplasts; Ferrous Compounds; Hydrogen Peroxide; Mitochondria; Models, Biological; Oxygen; Phenols; Plant Extracts; Plant Leaves; Signal Transduction; Sulfoxides; Xylenes

Peroxiredoxins (Prx) have recently moved into the focus of plant and animal research in the context of development, adaptation, and disease, as they function both in antioxidant defense by reducing a broad range of toxic peroxides and in redox signaling relating to the adjustment of cell redox and antioxidant metabolism. At-PrxII F is one of six type II Prx identified in the genome of Arabidopsis thaliana and the only Prx that is targeted to the plant mitochondrion. Therefore, it might be assumed to have functions similar to the human 2-Cys Prx (PRDX3) and type II Prx (PRDX5) and yeast 1-Cys Prx that likewise have mitochondrial localizations. This paper presents a characterization of PrxII F at the level of subcellular distribution, activity, and reductive regeneration by mitochondrial thioredoxin and glutaredoxin. By employing tDNA insertion mutants of A. thaliana lacking expression of AtprxII F (KO-AtPrxII F), it is shown that under optimal environmental conditions the absence of PrxII F is almost fully compensated for, possibly by increases in activity of mitochondrial ascorbate peroxidase and glutathione-dependent peroxidase. However, a stronger inhibition of root growth in KO-AtPrxII F seedlings as compared with wild type is observed under stress conditions induced by CdCl2 as well as after administration of salicylhydroxamic acid, an inhibitor of cyanide-insensitive respiration. Simultaneously, major changes in the abundance of both nuclear and mitochondria-encoded transcripts were observed. These results assign a principal role to PrxII F in antioxidant defense and possibly redox signaling in plants cells.

Topics: Adenosine Triphosphate; Amino Acid Sequence; Antioxidants; Arabidopsis; Ascorbic Acid; Blotting, Western; Cadmium; Cell Nucleus; Cell Proliferation; Cytosol; DNA; Dose-Response Relationship, Drug; Gene Expression Regulation, Plant; Genotype; Glutathione; Homeostasis; Immunohistochemistry; Mitochondria; Molecular Sequence Data; Oligonucleotide Array Sequence Analysis; Oxidation-Reduction; Oxidative Stress; Oxygen; Oxygen Consumption; Peroxidases; Peroxides; Peroxiredoxins; Phenols; Phenotype; Plant Roots; Recombinant Proteins; Reverse Transcriptase Polymerase Chain Reaction; Sequence Homology, Amino Acid; Subcellular Fractions; Sulfoxides; Time Factors; Tissue Distribution; Xylenes

Commonly used spectrophotometric methods for determining the extent of lipid peroxidation in animal tissue extracts, such as measurements of diene conjugation and thiobarbituric acid reactive substances (TBARS), have been criticized for their lack of specificity. This study shows that lipid hydroperoxides can be effectively quantified in animal tissue extracts using an assay based on the formation of a Fe(III)xylenol orange complex. Addition of H2O2, cumene hydroperoxides, or methanolic tissue extracts to an acidic reaction mixture containing 0.25 mM Fe(II) and 0.1 mM xylenol orange caused the formation of a broad Fe(III)xylenol orange complex absorbance peak at 560-580 nm with a corresponding decrease in the xylenol orange peak at 440 nm. Complex formation measured at 580 nm was saturable with both xylenol orange and Fe (II) concentration. Addition of ascorbic acid, GSH, and cysteine (0.3-5 mM) caused a saturable reduction of the Fe(III)xylenol orange complex. Formation of the Fe(III)xylenol orange complex was linear with the amount of tissue extract added. A significant correlation (r = 0.88, p < 0.005) existed between the xylenol orange method of estimating lipid peroxidation and the conventional TBARS assay in a series of animal tissues tested. The time course of increase in A580nm in tests using tissue extracts was typical of a free radical reaction; a lag phase was followed by a log phase. No increase in A580nm was observed up to 24 h when highly peroxidizable arachidonic acid was assayed. These results indicate that the formation of the Fe(III)xylenol orange complex reflects a chemical amplification of the original level of lipid hydroperoxides present in tissue extracts and that peroxidizable lipids do not influence the assay. The potential usefulness of the xylenol orange assay for comparative biochemical and toxicological studies of oxidative stress is discussed.

Topics: Animals; Ascorbic Acid; Chromatography, High Pressure Liquid; Cysteine; Ferric Compounds; Ferrous Compounds; Fluorescent Dyes; Glutathione; Indicators and Reagents; Kinetics; Lipid Peroxidation; Liver; Mice; Mice, Inbred Strains; Muscle, Skeletal; Phenols; Rats; Rats, Wistar; Sciuridae; Spectrophotometry; Sulfoxides; Thiobarbituric Acid Reactive Substances; Turtles; Xylenes