Cell cytotoxicity of sodium nitrite, sodium nitroprusside and Roussin's black salt against Trichomonas vaginalis

Abstract We have investigated the action of sodium nitrite and other nitrosyl complexes, such as sodium nitroprusside and Roussin's black salt, on the growth of metronidazole-sensitive and resistant strains of Trichomonas vaginalis and their hydrogenosomal enzymes. All three chemicals inhibited the growth of T. vaginalis : sodium nitrite at 8 mM, sodium nitroprusside at 1.2 mM and Roussin's black salt at 0.2 mM. Metronidazole-sensitive (KT9) and resistant (CDC85) isolates showed similar cytotoxicity against these molecules. Specific activities of pyruvate:ferredoxin oxidoreductase and hydrogenase and oxygen uptake rates were decreased in the T . vaginalis isolate treated with sodium nitrite and sodium nitroprusside. However, Roussin's black salt increased the specific activity of pyruvaterferredoxin oxidoreductase or hydrogenase in CDC85 or KT9 cells and increased the oxygen uptake rate in the KT9 isolate.     

Nitrosative stress induced cytotoxicity in Giardia intestinalis

AIMS: To investigate the antigiardial properties of the nitrosating agents: sodium nitrite, sodium nitroprusside and Roussin's black salt. METHODS AND RESULTS: Use of confocal laser scanning microscopy and flow cytometry indicated permeabilization of the plasma membrane to the anionic fluorophore, DiBAC4(3) [bis(1,3-dibutylbarbituric acid) trimethine oxonol]. Loss of plasma membrane electrochemical potential was accompanied by loss of regulated cellular volume control. Changes in ultrastructure revealed by electron microscopy and capacity for oxygen consumption, were also consequences of nitrosative stress. Roussin's black salt (RBS), active at micromolar concentrations was the most potent of the three agents tested. CONCLUSIONS: These multitargeted cytotoxic agents affected plasma membrane functions, inhibited cellular functions in Giardia intestinalis and led to loss of viability. SIGNIFICANCE AND IMPACT OF THE STUDY: Nitrosative damage, as an antigiardial strategy, may have implications for development of chemotherapy along with suggesting natural host defence mechanisms.     

Effects of sodium nitroprusside and nitrite on ouabaine-sensitive Na+, K(+)-ATPase activity of myometrium smooth muscle

Effective inhibiting effect of sodium nitroprusside and nitrite on Na+, K(+)-ATPase enzymatic activity of miometrium sarcolemma fraction was shown. Seeming Ki was of micromolar and submicromolar magnitudes. Investigations with sodium nitroprusside demonstrated an uncompetitive inhibition for ATP (growth of affinity for ATP and decrease of maximal velocity) and mixed inhibition for cations (decrease of maximal velocity and activation of constant for K+). Inhibitory effect of ouabain was reduced in the presence of sodium nitroprusside; ditiothreitol prevented enzyme inactivation by sodium nitroprusside. Kinetic analysis of experimental results using ouabain and ditiothreitol suggests chemical modification of enzyme sulfhydryl groups. Resistant component of Na+, K(+)-ATPase activity, which is sensitive to the action of detergent digitonine, was observed. In comparative investigations with postnucleus fraction stimulating actions of sodium nitroprusside, sodium nitrite, cGMP (more enhance) were shown. Methylene blue (soluble guanilate-cyclase inhibitor) prevented the activation of Na+, K(+)-ATPase activity by sodium nitrite. We suppose that the way of enzyme activation is prevalent in the condition of the moderate formation of nitric oxide and in the absence of hyper(over)production of reactive oxygen species.     

Nitric oxide inhibits calpain-mediated proteolysis of talin in skeletal muscle cells

We tested the hypothesis that nitric oxide caninhibit cytoskeletal breakdown in skeletal muscle cells by inhibitingcalpain cleavage of talin. The nitric oxide donor sodium nitroprusside prevented many of the effects of calcium ionophore onC₂C₁₂ muscle cells, including preventing talinproteolysis and release into the cytosol and reducing loss of vinculin,cell detachment, and loss of cellular protein. These results indicatethat nitric oxide inhibition of calpain protected the cells fromionophore-induced proteolysis. Calpain inhibitor I and a cell-permeablecalpastatin peptide also protected the cells from proteolysis,confirming that ionophore-induced proteolysis was primarily calpainmediated. The activity of m-calpain in a casein zymogram was inhibitedby sodium nitroprusside, and this inhibition was reversed bydithiothreitol. Previous incubation with the active site-targetedcalpain inhibitor I prevented most of the sodium nitroprusside-inducedinhibition of m-calpain activity. These data suggest that nitric oxideinhibited m-calpain activity via S-nitrosylation of the active sitecysteine. The results of this study indicate that nitric oxide produced endogenously by skeletal muscle and other cell types has the potential to inhibit m-calpain activity and cytoskeletal proteolysis.

     

Role of nitric oxide and mitochondria in control of firefly flash

In light-producing cells (photocytes) of the firefly light organ,mitochondria are clustered in the cell periphery, positionedbetween the tracheolar air supply and the oxygen-requiring bioluminescentreactants which are sequestered in more centrally-localizedperoxisomes. This relative positioning suggests that mitochondriacould control oxygen availability for the light reaction. Wehypothesized that active cellular respiration would make theinterior regions of the photocytes relatively hypoxic, and thatthe "on" signal for production of bioluminescence might dependon inhibition of mitochondrial oxygen consumption, which wouldallow delivered oxygen to pass through the peripheral mitochondrialzone to reach peroxisomes deep in the cell interior. We publishedrecently that exogenous NO induces bioluminescence in the intactfirefly; that NO mediates octopamine-induced bioluminescencein the dissected lantern, and that nitric oxide synthase isabundant in cells of the tracheolar system of the light organ.Additional experiments showed that nitric oxide gas (NO) inhibitsrespiration in isolated lantern mitochondria. Inhibition isreversed by bright light, and this inhibition is relieved whenthe light is turned off. Altogether, the results support theidea that NO triggers light production by reversible inhibitionof mitochondrial respiration in lantern cells, and probablyin tracheolar cells as well. The data also suggest that thelight of bioluminescence itself relieves NO inhibition thuscontributing to rapid on/off switching. While other mechanismsmay be in play, NO production that is directly related to neuralinput appears to have a key role in the oxygen gating that controlsflash communication signals.     

Inhibitor of Clostridium perfringens Formed by Heating Sodium Nitrite in a Chemically Defined Medium

An inhibitor of Clostridium perfringens formed when low levels of nitrite were autoclaved with a defined chemical medium. A systematic study of the medium revealed that only amino acids and mineral salts were involved in the production of this inhibitor, which was proven to be a toxic compound formed from cysteine, ferrous sulfate, and sodium nitrite. The inhibitor was compared to several known compounds. S-nitrosocysteine inhibited the test organism, but would not form in the test system in amounts large enough to explain the observed inhibition. Roussin red salt was unstable in the test system and therefore was not the inhibitor. Roussin black salt, which was also inhibitory, could form in sufficient amounts to explain the inhibition. A complex of cysteine, iron, and nitric oxide was detected in the autoclaved solution of cysteine, ferrous sulfate, and sodium nitrite; this cysteine complex did not appear to be inhibitory, however, at levels which could form in the autoclaved medium. The observed inhibition may have been due to the combined effects of sublethal concentrations of each compound.     

Endothelium-derived relaxing factor (nitric oxide) has protective actions in the stomach

The role that nitric oxide, an endothelium-derived relaxing factor, may play in the regulation of gastric mucosal defence was investigated by assessing the potential protective actions of this factor against the damage caused by ethanol in an ex vivo chamber preparation of the rat stomach. Topical application of glyceryl trinitrate and sodium nitroprusside, which have been shown to release nitric oxide, markedly reduced the area of 70% ethanol-induced hemorrhagic damage. Topical application of a 0.01% solution of authentic nitric oxide also significantly reduced the severity of mucosal damage. Pretreatment with indomethacin precluded the involvement of endogenous prostaglandins in the protective effects of these agents. The protective effects of NO were transient, since a delay of 5 minutes between NO administration and ethanol administration resulted in a complete loss of the protective activity. The protection against ethanol afforded by 10 micrograms/ml nitroprusside could be completely reversed by intravenous infusion of either 1% methylene blue or 1 mM hemoglobin, both of which inhibit vasodilation induced by nitric oxide. Intravenous infusion of 1% methylene blue significantly increased the susceptibility of the mucosa to damage induced by topical 20% ethanol. These results indicate that ethanol-induced gastric damage can be significantly reduced by nitric oxide. The mechanisms underlying the protective actions of nitric oxide are unclear, but may be related to its vasodilator or anti-aggregatory properties.     

Nitric oxide assisted hydrolysis of nitrophenylphosphate

In this study the use of sodium nitroprusside as nitric oxide donor in solutions was utilized. It has been established that maximum release of nitric oxide is achieved under irradiation by UV light at 254 nm. The synergistic effects of cobalt trimethylene diamine and nitroprusside towards the hydrolysis of nitrophenylphosphate under the above conditions for different cobalt to nitrophenylphosphate ratio were investigated. This study demonstrates that besides the effect of hydroxyl radicals, the direct interaction of nitric oxide with the phosphorous center also play a role in decontamination reactions of poorly biodegradable phosphate esters in natural waters, due to phototransformation.     

Nitric oxide and hypoxia exacerbate alcohol-induced mitochondrial dysfunction in hepatocytes

Chronic alcohol consumption results in hepatotoxicity, steatosis, hypoxia, increased expression of inducible nitric oxide synthase (iNOS) and decreased activities of mitochondrial respiratory enzymes. The impact of these changes on cellular respiration and their interaction in a cellular setting is not well understood. In the present study we tested the hypothesis that nitric oxide (NO)-dependent modulation of cellular respiration and the sensitivity to hypoxic stress is increased following chronic alcohol consumption. This is important since NO has been shown to regulate mitochondrial function through its interaction with cytochrome c oxidase, although at higher concentrations, and in combination with reactive oxygen species, can result in mitochondrial dysfunction. We found that hepatocytes isolated from alcohol-fed rats had decreased mitochondrial bioenergetic reserve capacity and were more sensitive to NO-dependent inhibition of respiration under room air and hypoxic conditions. We reasoned that this would result in greater hypoxic stress in vivo, and to test this, wild-type and iNOS(-/-) mice were administered alcohol-containing diets. Chronic alcohol consumption resulted in liver hypoxia in the wild-type mice and increased levels of hypoxia-inducible factor 1 α in the peri-venular region of the liver lobule. These effects were attenuated in the alcohol-fed iNOS(-/-) mice suggesting that increased mitochondrial sensitivity to NO and reactive nitrogen species in hepatocytes and iNOS plays a critical role in determining the response to hypoxic stress in vivo. These data support the concept that the combined effects of NO and ethanol contribute to an increased susceptibility to hypoxia and the deleterious effects of alcohol consumption on liver.     

Activation of a cytosolic ADP-ribosyltransferase by nitric oxide-generating agents

Sodium nitroprusside is a vasodilator and an inhibitor of platelet activation. It is thought that these effects are mediated by the spontaneous release of nitric oxide and stimulation of cytosolic guanylate cyclase. We have found that sodium nitroprusside (5-200 microM) greatly increased a cytosolic ADP-ribosyltransferase that ADP-ribosylates a soluble 39-kDa protein. This activity causes the mono-ADP-ribosylation of the 39-kDa protein, since digestion with snake venom phosphodiesterase releases 5'-AMP. This enzyme is present in platelets, brain, heart, intestine, liver, and lung. The effect of sodium nitroprusside is not related to stimulation of soluble guanylate cyclase and the production of cyclic GMP because cyclic GMP, dibutyryl cyclic GMP, and 8-bromo-cyclic GMP are ineffective. 3-Morpholinosydnonimine (commonly known as SIN-1) (20-1000 micrograms/ml), another compound that acts through the spontaneous formation of nitric oxide as does sodium nitroprusside, also stimulates ADP-ribosylation of the 39-kDa protein. Hemoglobin, which binds nitric oxide, inhibits sodium nitroprusside's activation of the cytosolic ADP-ribosyltransferase. These studies demonstrate a novel action of nitric oxide related to the activation of an endogenous ADP-ribosyltransferase. The physiological role of this ADP-ribosylation needs further exploration.     

Nitric oxide-induced S-nitrosylation of glyceraldehyde-3-phosphate dehydrogenase inhibits enzymatic activity and increases endogenous ADP-ribosylation.

Using conditions that produced chronic inflammation in rat liver, we were able to find a correlation between induction of nitric oxide production and inhibition of glyceraldehyde-3-phosphate dehydrogenase (GAPDH; EC 1.2.1.12). This enzyme is a tetramer composed of identical M(r) 37,000 subunits. The tetramer contains 16 thiol groups, four of which are essential for enzymatic activity. Our information indicates that four thiol groups are S-nitrosylated by exposure to authentic nitric oxide (NO) gas. Furthermore, NO decreased GAPDH activity while increasing its auto-ADP-ribosylation. Reduced nicotinamide adenine dinucleotide and dithiothreitol are required for the S-nitrosylation of GAPDH caused by the NO-generating compound sodium nitroprusside. Our results suggests that a new and important action of nitric oxide on cells is the S-nitrosylation and inactivation of GAPDH. S-Nitrosylation of GAPDH may be a key covalent modification of multiple regulatory consequences in chronic liver inflammation.     

Nitric oxide is toxic to melanocytes in vitro

Nitric oxide is a diffusible gaseous mediator generated from l-arginine by inducible and constitutive nitric oxide synthases. It has been associated with cytotoxic effects. Inflammatory cells and Langerhans cells can express the inducible form of nitric oxide synthase and produce large quantities of nitric oxide. The proximity of these cells to melanocytes could result in melanocyte cell death. We studied melanocyte susceptibility to nitric oxide using the nitric oxide donor compound sodium nitroprusside and nitric oxide released by the Langerhans like cell-line XS-52 following stimulation with lipopolysaccharide (LPS). Melanocyte lysis, quantified by chromium release in the presence of sodium nitroprusside was both time and concentration dependent. Co-culture of LPS-stimulated XS cells with melanocytes also resulted in melanocyte cell death. No cell death was observed when melanocytes alone were exposed to LPS. Melanocytes were killed even when the co-cultures were performed across Transwells in which there was no direct contact between XS cells and melanocytes. XS-induced melanocyte death was thus dependent on a diffusible factor consistent with nitric oxide. Cell death was markedly decreased in co-cultures performed in the presence of hemoglobin, a nitric oxide quencher. The possible role that nitric oxide may play in disorders associated with loss of pigmentation is discussed.     

Hydralazine decreases sodium nitroprusside-induced rat aortic ring relaxation and increased cGMP production by rat aortic myocytes

Association of hydralazine with nitrova-sodilators has long been known to be beneficial in the vasodilator treatment of heart failure. We previously found that hydralazine appeared to reduce the increase in cGMP induced by sodium nitroprusside in cultured rat aortic myocytes. In order to further explore this seemingly paradoxical interaction, we extended our initial observations in rat aortic myocytes and also determined the influence of hydralazine on sodium nitroprusside-induced relaxation of rat aortic rings. Hydralazine produced a concentration-dependent inhibition of sodium nitroprusside stimulation of cGMP production and caused a rightward shift of concentration-relaxation curves in aortic rings. A possible mechanism of the hydralazine-nitroprusside interaction could be the interference with bioactivation of the nitro-vasodilator to release nitric oxide. Recent evidence indicates that vascular NADH oxidase, an enzyme known to be inhibited by hydralazine, could be involved in this process. Accordingly, hydralazine was found to inhibit NADH oxidase activity in rat aortic myocytes at concentrations similar to those reducing sodium nitroprusside responses. It was concluded that antagonism of sodium nitroprusside action by hydralazine could be a consequence of interference with bioactivation of the former, apparently through inhibition of vascular NADH oxidase.     

NO对金丝桃悬浮细胞生长及金丝桃素生物合成的促进作用研究

一氧化氮 (NO)是近年来发现的一种新型植物信号分子。以硝普钠 (Sodiumnitroprusside ,SNP)为一氧化氮 (NO)的供体 ,研究外源NO对金丝桃悬浮细胞生长及金丝桃素生物合成的影响。试验结果表明 ,金丝桃悬浮细胞在含 0 5和 15 0mmol LSNP的培养基中培养 2 0d后 ,细胞的干重分别为对照组的 140%和50% ;细胞中金丝桃素的含量分别为对照组的 98%和210%。试验结果表明 ,低浓度SNP处理有利于金丝桃悬浮细胞生长 ,而高浓度SNP可以促进金丝桃素的合成。在细胞培养初期 (0d)加入 0.5mmol LSNP并在指数生长后期 (14d)加入15.0mmol LSNP的金丝桃悬浮细胞在培养 2.5d后 ,细胞的干重和金丝桃素的含量分别为对照组的1.4和1.8倍 ,金丝桃素的产量达15.2mg/L ,比对照高3.2倍。SNP对金丝桃悬浮细胞生长及金丝桃素含量的影响可以被NO专一性淬灭剂CPITO(2-4-carboxyphenyl-4 ,4 ,5 ,5-tetramethylimidazoline-1-oxyl-3-oxide)所抑制,说明SNP是通过其分解产物NO影响细胞生长和金丝桃素的合成。试验结果同时表明,在15.0mmol/L的SNP处理下,金丝桃悬浮细胞中的苯丙氨酸解氨酶(PAL)的活性显著升高,推测NO可能通过触发金丝桃悬浮细胞的防卫反应,激活了细胞中金丝桃素的生物合成途径。     

Nitric oxide inhibits cardiac energy production via inhibition of mitochondrial creatine kinase

Nitric oxide biosynthesis in cardiac muscle leads to a decreased oxygen consumption and lower ATP synthesis. It is suggested that this effect of nitric oxide is mainly due to the inhibition of the mitochondrial respiratory chain enzyme, cytochrome c oxidase. However, this work demonstrates that nitric oxide is able to inhibit soluble mitochondrial creatine kinase (CK), mitochondrial CK bound in purified mitochondria, CK in situ in skinned fibres as well as the functional activity of mitochondrial CK in situ in skinned fibres. Since mitochondrial isoenzyme is functionally coupled to oxidative phosphorylation, its inhibition also leads to decreased sensitivity of mitochondrial respiration to ADP and thus decreases ATP synthesis and oxygen consumption under physiological ADP concentrations.     

Nitrite modulates bacterial antibiotic susceptibility and biofilm formation in association with airway epithelial cells

Pseudomonas aeruginosa is the major pathogenic bacteria in cystic fibrosis and other forms of bronchiectasis. Growth in antibiotic-resistant biofilms contributes to the virulence of this organism. Sodium nitrite has antimicrobial properties and has been tolerated as a nebulized compound at high concentrations in human subjects with pulmonary hypertension; however, its effects have not been evaluated on biotic biofilms or in combination with other clinically useful antibiotics. We grew P. aeruginosa on the apical surface of primary human airway epithelial cells to test the efficacy of sodium nitrite against biotic biofilms. Nitrite alone prevented 99% of biofilm growth. We then identified significant cooperative interactions between nitrite and polymyxins. For P. aeruginosa growing on primary CF airway cells, combining nitrite and colistimethate resulted in an additional log of bacterial inhibition compared to treating with either agent alone. Nitrite and colistimethate additively inhibited oxygen consumption by P. aeruginosa. Surprisingly, whereas the antimicrobial effects of nitrite in planktonic, aerated cultures are nitric oxide (NO) dependent, antimicrobial effects under other growth conditions are not. The inhibitory effect of nitrite on bacterial oxygen consumption and biofilm growth did not require NO as an intermediate as chemically scavenging NO did not block growth inhibition. These data suggest an NO-radical independent nitrosative or oxidative inhibition of respiration. The combination of nebulized sodium nitrite and colistimethate may provide a novel therapy for chronic P. aeruginosa airway infections, because sodium nitrite, unlike other antibiotic respiratory chain “poisons,” can be safely nebulized at high concentration in humans.     

Neither nitrite nor nitric oxide mediate toxic effects of nitroglycerin on mitochondria

It is commonly accepted that the major effect of nitroglycerin (NG) is realized through the release of nitric oxide (NO) catalyzed by aldehyde dehydrogenase-2 (ALDH2). In addition, it has been shown that NG inhibits mitochondrial respiration. The aim of this study was to clarify whether NG-mediated inhibition of mitochondrial respiration is mediated by NO. In rat liver mitochondria, NG inhibited complex-I-dependent respiration and induced reactive oxygen species (ROS) production, preferentially at complex I. Both effects were insensitive to chloral hydrate, an ALDH2 inhibitor. Nitrite, an NG intermediate, had no influence on either mitochondrial respiration or the production of ROS. NO inhibited preferentially complex I but did not elevate ROS production. Hemoglobin, an NO scavenger, and blue light had contrary effects on mitochondria inhibited by NO or NG. In summary, our data suggest that although NG induces vasodilatation via NO release, it causes mitochondrial dysfunction via an NO-independent pathway.     

Properties of purified soluble guanylate cyclase activated by nitric oxide and sodium nitroprusside

Highly purified rat lung soluble guanylate cyclase was activated with nitric oxide or sodium nitroprusside and the degree of activation varied with incubation conditions. With Mg2+ as the action cofactor, about 2- to 8-fold activation was observed with nitric oxide or sodium nitroprusside alone. Markedly enhanced activation (20-40 fold) was observed when 1 muM hemin added to the enzyme prior to exposure to the activating agent. The activation with hemin and sodium nitroprusside was prevented in a dose-dependent manner by sodium cyanide. The level activation was also increased by the addition of 1 mM dithiothreitol, but unlike hemin which had no effect on basal enzyme activity, dithiothreitol led to a considerable increase in basal activity. Activated guanylate cyclase decayed to basal activity within one hour at 2 degrees C and the enzyme could be reactivated upon re-exposure to nitroprusside or nitric oxide. Under basal conditions, Michaelis-Menten kinetics were observed, with a Km for GTP of 140 muM with Mg2+ cofactor. Following activation with nitroprusside or nitric oxide, curvilinear Eadie-Hofstee transformations of kinetic data were observed, with Km's of 22 MuM and 100 MuM for Mg-GTP. When optimal activation (15-40 fold) was induced by the addition of hemin and nitroprusside, multiple Km's were also seen with Mg-GTP and the high affinity form was predominant (22 MuM). Similar curvilinear Eadie-Hofstee transformations were observed with Mn2+ as the cation cofactor. These data suggest that multiple GTP catalytic sites are present in activated guanylate cyclase, or alternatively, multiple populations of enzyme exist.     

Role of an inducible single-domain hemoglobin in mediating resistance to nitric oxide and nitrosative stress in Campylobacter jejuni and Campylobacter coli

Campylobacter jejuni expresses two hemoglobins, each of which exhibits a heme pocket and structural signatures in common with vertebrate and plant globins. One of these, designated Cgb, is homologous to Vgb from Vitreoscilla stercoraria and does not possess the reductase domain seen in the flavohemoglobins. A Cgb-deficient mutant of C. jejuni was hypersensitive to nitrosating agents (S-nitrosoglutathione [GSNO] or sodium nitroprusside) and a nitric oxide-releasing compound (spermine NONOate). The sensitivity of the Cgb-deficient mutant to methyl viologen, hydrogen peroxide, and organic peroxides, however, was the same as for the wild type. Consistent with the protective role of Cgb against NO-related stress, cgb expression was minimal in standard laboratory media but strongly and specifically induced after exposure to nitrosative stress. In contrast, the expression of Cgb was independent of aeration and the presence of superoxide. In the absence of preinduction by exposure to nitrosative stress, no difference was seen in the degree of respiratory inhibition by NO or the half-life of the NO signal when cells of the wild type and the cgb mutant were compared. However, cells expressing GSNO-upregulated levels of Cgb exhibited robust NO consumption and respiration that was relatively NO insensitive compared to the respiration of the cgb mutant. Based on similar studies in Campylobacter coli, we also propose an identical role for Cgb in this closely related species. We conclude that, unlike the archetypal single-domain globin Vgb, Cgb forms a specific and inducible defense against NO and nitrosating agents.     

NO-dependent phototoxicity of Roussin's black salt against cancer cells.

A metal-nitrosyl complex, Roussin's black salt (RBS), releases nitric oxide after illumination. Approximately 3.7 NO molecules were released from one RBS molecule. Both short- and long-term effects of photogenerated NO on the two neoplastic cell lines: human (SK-MEL188) and mouse (S91) have been investigated. Exogenous NO from RBS was toxic to cells in a dose-dependent manner. Apoptotic damage predominates in the response to the injury, as shown by TUNEL assay. NO and its short-lived metabolites, but not other RBS photoproducts, are responsible for cellular death. RBS in dark was toxic to cells at concentrations above 1 microM. This relatively high cytotoxicity of RBS in the dark prevents its application as a systemic anticancer agent in vivo, unless it is applied locally.