Dual-function of thiocyanate on nitrite-induced formation of reactive nitrogen oxide species in human oral cavity: inhibition under neutral and enhancement under acidic conditions

Salivary nitrate is reduced to nitric oxide (NO) via nitrite in the human oral cavity. The nitrite and NO formed can be transformed to reactive nitrogen oxide species (RNOS). In this investigation, RNOS formed in mixed whole saliva and its fractions were detected by the oxidation of aminophenyl fluorescein (APF) and the transformation of 3-amino-4-monomethylamino-2',7'-difluorofluorecein (DAF-FM) to its triazol form (DAF-FMT). Nitrite-induced oxidation of APF and formation of DAF-FMT increased as pH was decreased from 7 to 5 and SCN^- inhibited the oxidation of APF and the formation of DAF-FMT around neutral pH and enhanced at pH about 5. The SCN^--dependent inhibition was due to the suppression of salivary peroxidase and the enhancement was due to the formation of NOSCN from HNO₂ and SCN^-. It is deduced that the increase in the concentrations of nitrite and H⁺ in the oral cavity may result in the enhanced formation of RNOS.     

Dual-function of thiocyanate on nitrite-induced formation of reactive nitrogen oxide species in human oral cavity: Inhibition under neutral and enhancement under acidic conditions

Salivary nitrate is reduced to nitric oxide (NO) via nitrite in the human oral cavity. The nitrite and NO formed can be transformed to reactive nitrogen oxide species (RNOS). In this investigation, RNOS formed in mixed whole saliva and its fractions were detected by the oxidation of aminophenyl fluorescein (APF) and the transformation of 3-amino-4-monomethylamino-2′,7′-difluorofluorecein (DAF-FM) to its triazol form (DAF-FMT). Nitrite-induced oxidation of APF and formation of DAF-FMT increased as pH was decreased from 7 to 5 and SCN^? inhibited the oxidation of APF and the formation of DAF-FMT around neutral pH and enhanced at pH about 5. The SCN^?-dependent inhibition was due to the suppression of salivary peroxidase and the enhancement was due to the formation of NOSCN from HNO₂ and SCN^?. It is deduced that the increase in the concentrations of nitrite and H⁺ in the oral cavity may result in the enhanced formation of RNOS.     

Production of nitric oxide-derived reactive nitrogen species in human oral cavity and their scavenging by salivary redox components

Nitrite is reduced to nitric oxide (NO) in the oral cavity. The NO generated can react with molecular oxygen producing reactive nitrogen species. In this study, reduction of nitrite to NO was observed in bacterial fractions of saliva and whole saliva. Formation of reactive nitrogen species from NO was detected by measuring the transformation of 4,5-diaminofluorescein (DAF-2) to triazolfluorescein (DAF-2T). The transformation was fast in bacterial fractions but slow in whole saliva. Salivary components such as ascorbate, glutathione, uric acid and thiocyanate inhibited the transformation of DAF-2 to DAF-2T in bacterial fractions without affecting nitrite-dependent NO production. The inhibition was deduced to be due to scavenging of reactive nitrogen species, which were formed from NO, by the above reagents. The transformation of DAF-2 to DAF-2T was faster in bacterial fractions and whole saliva which were prepared 1–4?h after tooth brushing than those prepared immediately after toothbrushing. Increase in the rate as a function of time after toothbrushing seemed to be due to the increase in population of bacteria which could reduce nitrite to NO. The results obtained in this study suggest that reactive nitrogen species derived from NO are continuously formed in the oral cavity and that the reactive nitrogen species are effectively scavenged by salivary redox components in saliva but the scavenging is not complete.     

Production of nitric oxide-derived reactive nitrogen species in human oral cavity and their scavenging by salivary redox components

Nitrite is reduced to nitric oxide (NO) in the oral cavity. The NO generated can react with molecular oxygen producing reactive nitrogen species. In this study, reduction of nitrite to NO was observed in bacterial fractions of saliva and whole saliva. Formation of reactive nitrogen species from NO was detected by measuring the transformation of 4,5-diaminofluorescein (DAF-2) to triazolfluorescein (DAF-2T). The transformation was fast in bacterial fractions but slow in whole saliva. Salivary components such as ascorbate, glutathione, uric acid and thiocyanate inhibited the transformation of DAF-2 to DAF-2T in bacterial fractions without affecting nitrite-dependent NO production. The inhibition was deduced to be due to scavenging of reactive nitrogen species, which were formed from NO, by the above reagents. The transformation of DAF-2 to DAF-2T was faster in bacterial fractions and whole saliva which were prepared 1-4 h after tooth brushing than those prepared immediately after toothbrushing. Increase in the rate as a function of time after toothbrushing seemed to be due to the increase in population of bacteria which could reduce nitrite to NO. The results obtained in this study suggest that reactive nitrogen species derived from NO are continuously formed in the oral cavity and that the reactive nitrogen species are effectively scavenged by salivary redox components in saliva but the scavenging is not complete.     

Aggrecan degradation in chondrocytes is mediated by reactive oxygen species and protected by antioxidants

Reactive oxygen species (ROS) are implicated in aging of cartilage and in the pathogenesis of osteoarthritis. However, the biological role of chondrocytes-derived ROS has not been elucidated. An in-vitro model was developed to study the role of chondrocyte-derived ROS in cartilage matrix degradation. The primary articular chondrocytes were cultured and the aggrecan matrix was radiolabeled with 35-sulfate. The labeled aggrecan matrix was washed to remove unincorporated label and chondrocytes were returned to serum free balanced salt solution. The cell-monolayer-matrix sensitivity to oxidative damage due to either hydrogen peroxide or glucose oxidase was established by monitoring the release of labeled aggrecan into the medium. Lipopolysaccharide (LPS) treatment of chondrocytemonolayer enhanced the release of labeled aggrecan. Catalase significantly prevented the release of labeled aggrecan in LPS-chondrocyte cultures, suggesting a role for chondrocyte-derived hydrogen peroxide in aggrecan degradation. Superoxide dismutase or boiled catalase had no such inhibitory effect. The effect of several antioxidants on LPS-chondrocyte-dependent aggrecan degradation was examined. Hydroxyl radical scavengers (mannitol and thiourea) significantly decreased aggrecan degradation. A spin trapping agent N-tert-butyl-phenylnitrone (but not its inactive analog tert-butyl-phenylcarbonate) significantly decreased aggrecan degradation. Butylated hydroxytoluene also inhibited aggrecan degradation, whereas the other lipophilic antioxidant tested, propyl gallate, had a marked dose-dependent inhibitory effect. These data indicate that general antioxidants, hydroxyl radical scavengers, antioxidant vitamins, iron chelating agents, lipophilic antioxidants, and spin trapping agents can influence chondrocyte-dependent aggrecan degradation. These studies support the role of a chondrocyte-dependent oxidative mechanism in aggrecan degradation and indicate that antioxidants can prevent matrix degradation and therefore may have a preventive or therapeutic value in arthritis. The enhancement of oxidative activity in chondrocytes and its damaging effect on matrix may be an important mechanism of matrix degradation in osteoarthritis.     

Mechanism of teratogenesis: electron transfer, reactive oxygen species, and antioxidants

Teratogenesis has been a topic of increasing interest and concern in recent years, generating controversy in association with danger to humans and other living things. A veritable host of chemicals is known to be involved, encompassing a wide variety of classes, both organic and inorganic. Contact with these chemicals is virtually unavoidable due to contamination of air, water, ground, food, beverages, and household items, as well as exposure to medicinals. The resulting adverse effects on reproduction are numerous. There is uncertainty regarding the mode of action of these chemicals, although various theories have been advanced, e.g., disruption of the central nervous system (CNS), DNA attack, enzyme inhibition, interference with hormonal action, and insult to membranes, proteins, and mitochondria. This review provides extensive evidence for involvement of oxidative stress (OS) and electron transfer (ET) as a unifying theme. Successful application of the mechanistic approach is made to all of the main classes of toxins, in addition to large numbers of miscellaneous types. We believe it is not coincidental that the vast majority of these substances incorporate ET functionalities (quinone, metal complex, ArNO2, or conjugated iminium) either per se or in metabolites, potentially giving rise to reactive oxygen species (ROS) by redox cycling. Some categories, e.g., peroxides and radiation, appear to generate ROS by non-ET routes. Other mechanisms are briefly addressed; a multifaceted approach to mode of action appears to be the most logical. Our framework should increase understanding and contribute to preventative measures, such as use of antioxidants.     

Determination of reactive oxygen and nitrogen species in rat aorta using the dichlorofluorescein assay

A method for the determination of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in macroscopic sections of vessels has been developed on the basis of the dichlorofluorescein (DCF) assay. DCF was measured by fluorescence in extracts of vessels. The main artifact of the method is the oxidation of dichlorodihydrofluorescein (DCFH₂) which is released from vessels together with DCF during the extraction procedure. This problem was resolved by decreasing pH during the extraction. The optimal conditions and the time for aorta incubation with DCFH₂-DA and for the extraction of DCF from aorta have been determined. The ROS/RNS production in different aorta segments and the dependence of ROS/RNS production on rat age have been studied. It was shown that thoracic aorta sections produced the same amounts of ROS/RNS and the intermediate between the thoracic and the abdominal aorta part produced ROS and RNS by 14% more than the thoracic aorta. It was found that ROS/RNS production in aorta increases with rat age: the doubling time of ROS/RNS production rate is 113 days from birth.     

Influence of antioxidants on NO-dependent induction of heme oxygenase-1 in U937 monocytes

Thiol antioxidants are known to inhibit the nitric oxide-dependent induction of the hemoxygenase-1 gene (HOX-1). To estimate the degree to which the inhibitory effect of thiol antioxidants is accounted for by them scavenging oxidized NO derivatives or their precursors, the reactive oxygen and nitrogen species (ROS and RNS), we studied the inhibitory effect of nonthiol antioxidants: dimethyl sulfoxide, dimethylthiourea, sodium salicylate, sodium formate, uric acid, catalase, and superoxide dismutase. Partial inhibition of NO-dependent HOX-1 induction was observed in the presence of the nonpolar HO scavengers dimethyl sulfoxide and dimethylthiourea. The antioxidants which selectively bind other ROS had no effect on HOX-1 expression. To reveal the role of RNS in NO-dependent HOX-1 induction, cells were treated with the NO-generating compound DPTA-NO in the presence of 2-phenyl-4,4,5,5,-tetramethylimidazole-1-oxyl 3 oxide (PTIO), which oxidizes NO to NO₂. PTIO proved to significantly enhance NO-dependent HOX-1 induction. Thiol antioxidants completely inhibited the stimulating effect of PTIO, which is evidence that their inhibitory effect is explained by RNS scavenging. The results of this study indicate that antioxidants can be used to modulate the cell response to NO.Translated from Molekulyarnaya Biologiya, Vol. 39, No. 1, 2005, pp. 89–95.Original Russian Text Copyright © 2005 by Litvinov, Prasolov, Bouton, Drapier, Turpaev.     

Prolongation of oxidative stress by long-lived reactive protein species induced by X-ray radiation and their genotoxic action

Abstract

The formation of long-lived reactive protein species of bovine serum albumin (BSA), ovalbumin, casein and casein hydrolyzate with a half-life of 3–5 hours was shown using chemiluminescence induced by X-ray radiation. It was found that long-lived reactive protein species are capable of generating reactive oxygen species (ROS) (H₂O₂, OH^?, HO₂^{?, 1}O₂) in the aquatic environment over a long period of time in vitro. The interaction of X-ray-irradiated BSA with DNA in vitro led to the formation of 8-oxoguanine (8-oxo-7,8-dihydroguanine), a biomarker of oxidative damage to DNA. Some natural antioxidants are effective scavengers of ROS (inosine, tryptophan, methionine and ascorbate). They protect DNA from the action of long-lived reactive protein species leading to ROS generation and the formation of 8-oxoguanine. The intravenous injection of X-ray radiation-induced, long-lived reactive protein species to rats, as well as the peroral and intraperitoneal administration of these products to mice, gave rise to cytogenetic injuries in the cells of their red bone marrow through the formation of micronuclei in polychromatophilic erythrocytes. The administration of the same natural antioxidants used for in vitro experiments soon after irradiation made it possible to effectively eliminate the genotoxic action of oxidative stress caused by radiation-induced, long-lived reactive protein species. Our data represent clear evidence that the oxidative damage to proteins induced by X-rays is directly involved in the induction of a response to DNA damage in rodents.     

叶绿体光合代谢中活性氧的产生与清除

有氧代谢不可避免产生活性氧(ROS),叶绿体的PSI和PSII反应中心均是ROS产生的主要位点。叶绿体产生的ROS主要有超氧阴离子(O2-)、过氧化氢(H2O2)、羟自由基(.OH)和单线氧(1O2),其中在PSI产生的O2-将进一步产生H2O2和.OH,而1O2产生在PSII。正常生理代谢条件下,叶绿体内抗氧化系统和光能吸收利用的调节保持活性氧产生和消灭的平衡,不会影响植物的正常生理功能。     

Age-dependent change in reactive oxygen species and nitric oxide generation by rat alveolar macrophages

Immunosenescence is an age-associated dysregulation of the immune function, which contributes to increased susceptibility to disease in the elderly. Alveolar macrophages (AM) are known phagocytes that generate reactive oxygen species (ROS) and nitric oxide (NO), essential mediators for host defence. We studied phagocytosis, ROS and NO production in AM obtained from young, adult and senescent rats (1-2, 9-12 and 18-24 months old, respectively) after exposure to lipopolysaccharide (LPS, 0.1-10 microg mL(-1)), 12-O-tetradecanoylphorbol 13-acetate (TPA, 0.1 microg mL(-1)) or LPS + TPA in culture. Phagocytosis was significantly lower in control AM from adult rats than in AM from young animals. Nevertheless, AM from adult animals pretreated with LPS exhibited higher phagocytic capacity than AM from younger animals. ROS was identified by the NBT test at single cell level and quantified by automated image analysis. When TPA was added to all three populations, AM from adult and senescent animals responded more than AM from young animals. All LPS-stimulated AM produce more NO than controls. However, NO production increased three-, four- and two-fold in young, adult and senescent animals, respectively. Our results demonstrate that AM from young, adult and senescent animals display differential responsiveness to inflammatory mediators. Therefore, aging processes markedly affect AM metabolic functions and may further compromise the lung immune defence response, increasing adverse long-term health effects.     

Functional complementation in yeast reveals a protective role of chloroplast 2-Cys peroxiredoxin against reactive nitrogen species

The importance of nitric oxide (NO) as a signaling molecule to various plant physiological and pathophysiological processes is becoming increasingly evident. However, little is known about how plants protect themselves from nitrosative and oxidative damage mediated by NO and NO-derived reactive nitrogen species (RNS). Peroxynitrite, the product of the reaction between NO and superoxide anion, is considered to play a central role in RNS-induced cytotoxicity, as a result of its potent ability to oxidize diverse biomolecules. Employing heterologous expression in bacteria and yeast, we investigated peroxynitrite-scavenging activity in plants of 2-Cys peroxiredoxin (2CPRX), originally identified as a hydroperoxide-reducing peroxidase that is ubiquitously distributed among organisms. The putative mature form of a chloroplast-localized 2CPRX from Arabidopsis thaliana was overproduced in Escherichia coli as an amino-terminally hexahistidine-tagged fusion protein. The purified recombinant 2CPRX, which was catalytically active as peroxidase, efficiently prevented the peroxynitrite-induced oxidation of a sensitive compound. We also examined in vivo the ability of the Arabidopsis 2CPRX to complement the 2CPRX deficiency of a Saccharomyces cerevisiae mutant. Functional expression in the mutant strain of the Arabidopsis 2CPRX not only increased cellular tolerance to hydrogen peroxide, but also complemented the hypersensitive growth defect induced by nitrite-mediated cytotoxicity. The complemented cells significantly enhanced the capacity to reduce RNS-mediated oxidative damages. The results presented here demonstrate a new role of plant 2CPRX as a critical determinant of the resistance to RNS, and support the existence of a plant enzymatic basis for RNS metabolism.     

Effect of antioxidants on chemiluminescence produced by reactive oxygen species

Luminol chemiluminescence was used to evaluate the scavenging of superoxide, hydroxyl and alkoxy radicals by four antioxidants: dipyridamole, diethyldithiocarbamic acid, (+)catechin, and ascorbic acid. Different concentrations of these compounds were compared with well-known oxygen radical scavengers in their capacity to inhibit the chemiluminescence produced in the reaction between luminol and specific oxygen radicals. Hydroxyl radicals were generated using the Fenton reaction and these produced chemiluminescence which was inhibited by diethyldithiocarbamate. Alkoxy radicals were generated using the reaction of tert-butyl hydroperoxide and ferrous ion and produced chemiluminescence which was inhibited equally by all of the compounds tested. For the determination of superoxide scavengers we describe a new, simple, economic, and rapid chemiluminescence method consisting of the reaction between luminol and horseradish peroxidase (HRP). With this method it was found that 40 nmol/l dipyridamole, 0.18 μmol/l ascorbic acid, 0.23 μmol/l (+)catechin, and 3 μmol/l diethyldithiocarbamic acid are equivalent to 3.9 ng/ml superoxide dismutase (specific scavenger of superoxide) in causing the same degree of chemiluminescence inhibition. These results not only indicated that the antioxidative properties of these compounds showed different degrees of effectiveness against a particular radical but also that they may exert their action against more than one radical.     

Cell signaling by reactive nitrogen and oxygen species in atherosclerosis

The production of reactive oxygen and nitrogen species has been implicated in atherosclerosis principally as means of damaging low-density lipoprotein that in turn initiates the accumulation of cholesterol in macrophages. The diversity of novel oxidative modifications to lipids and proteins recently identified in atherosclerotic lesions has revealed surprising complexity in the mechanisms of oxidative damage and their potential role in atherosclerosis. Oxidative or nitrosative stress does not completely consume intracellular antioxidants leading to cell death as previously thought. Rather, oxidative and nitrosative stress have a more subtle impact on the atherogenic process by modulating intracellular signaling pathways in vascular tissues to affect inflammatory cell adhesion, migration, proliferation, and differentiation. Furthermore, cellular responses can affect the production of nitric oxide, which in turn can strongly influence the nature of oxidative modifications occurring in atherosclerosis. The dynamic interactions between endogenous low concentrations of oxidants or reactive nitrogen species with intracellular signaling pathways may have a general role in processes affecting wound healing to apoptosis, which can provide novel insights into the pathogenesis of atherosclerosis.     

Insulin resistance and diabetes in hyperthyroidism: a possible role for oxygen and nitrogen reactive species

In addition to insulin, glycemic control involves thyroid hormones. However, an excess of thyroid hormone can disturb the blood glucose equilibrium, leading to alterations of carbohydrate metabolism and, eventually, diabetes. Indeed, experimental and clinical hyperthyroidism is often accompanied by abnormal glucose tolerance. A common characteristic of hyperthyroidism and type 2 diabetes is the altered mitochondrial efficiency caused by the enhanced production of reactive oxygen and nitrogen species. It is known that an excess of thyroid hormone leads to increased oxidant production and mitochondrial oxidative damage. It can be hypothesised that these species represent the link between hyperthyroidism and development of insulin resistance and diabetes, even though direct evidence of this relationship is lacking. In this review, we examine the literature concerning the effects of insulin and thyroid hormones on glucose metabolism and discuss alterations of glucose metabolism in hyperthyroid conditions and the cellular and molecular mechanisms that may underline them.     

Modulation of mitochondrial membrane potential and reactive oxygen species production by copper in astrocytes

In monolayers of cultured rat astrocytes a number of agents that induce oxidative stress act synergistically with exposure to copper leading to rapid depolarization of the mitochondrial membrane potential (Psi m) and increased reactive oxygen species (ROS) production. Copper sensitized astrocytes to the action of menadione, an intracellular generator of superoxide anion radical, exogenous hydrogen peroxide (H2O2) and rotenone, an inhibitor of mitochondrial electron transport chain complex I. However, significant differences were observed in the ability to modulate the copper-enhanced oxidative stress depending on which stressor was used. The inhibitor of mitochondrial permeability transition cyclosporin A attenuated the effect of copper and rotenone, but had no protective action in the case of H2O2/copper and menadione/copper combinations. The H2O2 scavenger pyruvate was effective at protecting mitochondria against damage associated with the combined exposure to H2O2/copper and menadione/copper but not to the rotenone/copper combination. The antioxidant Trolox was ineffective at protecting against any of these actions and indeed had a damaging effect when combined with copper. The membrane-permeable copper chelator neocuproine combined with sensitizing concentrations of menadione caused a decrease in Psi m, mimicking the action of copper. Penicillamine, a membrane-impermeable copper chelator, was effective at reducing copper sensitization. Endogenous copper, mobilized during periods of oxidative stress, may play a role in the pathophysiology of brain injury. Our results suggest that this might be particularly dangerous in dysfunctional conditions in which the mitochondrial electron transport chain is compromised.     

Ontogenic profile of some antioxidants and lipid peroxidation in human placental and fetal tissues

Reactive oxygen species (ROS) pose a serious threat to maternal and fetal health during pregnancy. However, there is little information on the oxidative damage caused by ROS and its protection during prenatal life. The present study highlights the status of various antioxidants in human placental and fetal tissues at different phases of gestation. The activity profile of scavenging enzymes, superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase as well as the concentrations of non-enzymatic antioxidants, ascorbic acid, -tocopherol, bilirubin and glutathione have been determined in human placental whole homogenate, placental brush border membrane and fetal liver over gestational periods ranging from 6 weeks of pregnancy till birth. The ontogenic profile of lipid peroxidation, a marker of oxidative damage has also been investigated in the feto-placental system. Catalase, superoxide dismutase and glutathione reductase activities increased significantly, but glutathione peroxidase activity remained almost the same throughout development. Except - tocopherol and bilirubin, the concentrations of other non-enzymic scavengers followed a significant increasing trend with advancement of pregnancy. Results indicate that there is gradual suppression of lipoperoxide formation with the progress of gestation to protect the fetus against oxygen toxicity.     

Global regulation of reactive oxygen species scavenging genes in alfalfa root and shoot under gradual drought stress and recovery

Reactive oxygen species (ROS) production and scavenging in plants under drought stress have been studied intensively in recent years. Here we report a global analysis of gene expression for the major ROS generating and scavenging proteins in alfalfa root and shoot under gradual drought stress followed by one-day recovery. Data from two alfalfa varieties, one drought tolerant and one drought sensitive, were compared and no qualitative differences in ROS gene regulation between the two were found. Conserved, tissue-specific patterns of gene expression in response to drought were observed for several ROS-scavenging gene families, including ascorbate peroxidase, monodehydroascorbate reductase, and peroxiredoxin. In addition, differential gene expression within families was observed. Genes for the ROS-generating enzyme, NADPH oxidase were generally induced under drought, while those for glycolate oxidase were repressed. Among the ROS-scavenging protein genes, Ferritin, Cu/Zn superoxide dismutase (SOD), and the majority of the glutathione peroxidase family members were induced under drought in both roots and shoots of both alfalfa varieties. In contrast, Fe-SOD, CC-type glutaredoxins, and thoiredoxins were downregulated.