The effect of Nano-liposomal sodium nitrite on smooth muscle cell growth in a tissue-engineered small-diameter vascular graft.

BACKGROUND: Nitric oxide is a chemical agent produced by endothelial cells in a healthy blood vessel, inhibiting the overgrowth of vascular smooth muscle cells and regulating vessel tone. Liposomes are biocompatible and biodegradable drug carriers with a similar structure to cell bilayer phospholipid membrane that can be used as useful nitric oxide carriers in vascular grafts. METHOD: Using a custom-designed apparatus, the sheep carotid arteries were decellularized while still maintaining important components of the vascular extracellular matrix (ECM), allowing them to be used as small-diameter vascular grafts. A chemical signal of sodium nitrite was applied to control smooth muscle cells' behavior under static and dynamic cell culture conditions. The thin film hydration approach was used to create nano-liposomes, which were then used as sodium nitrite carriers to control the drug release rate and enhance the amount of drug loaded into the liposomes. RESULTS: The ratio of 80:20:2 for DPPC: Cholesterol: PEG was determined as the optimum formulation of the liposome structure with high drug encapsulation efficiency (98%) and optimum drug release rate (the drug release rate was 40%, 65%, and 83% after 24, 48, and 72 h, respectively). MTT assay results showed an improvement in endothelial cell proliferation in the presence of nano-liposomal sodium nitrite (LNS) at the concentration of 0.5 µg/mL. Using a suitable concentration of liposomal sodium nitrite (0.5 µg/mL) put onto the constructed scaffold resulted in the controllable development of smooth muscle cells in the experiment. The culture of smooth muscle cells in a pulsatile perfusion bioreactor indicated that in the presence of synthesized liposomal sodium nitrite, the overgrowth of smooth muscle cells was inhibited in dynamic cell culture conditions. The mechanical properties of ECM graft were measured, and a multi-scale model with an accuracy of 83% was proposed to predict mechanical properties successfully. CONCLUSION: The liposomal drug-loaded small-diameter vascular graft can prevent the overgrowth of SMCs and the formation of intimal hyperplasia in the graft. Aside from that, the effect of LNS on endothelial has the potential to stimulate endothelial cell proliferation and re-endothelialization.

Variation in glucose metabolism under acidified sodium nitrite mediated nitrosative stress in Saccharomyces cerevisiae.

AIMS: The work aimed to understand the important changes during glucose metabolism in Saccharomyces cerevisiae under acidified sodium nitrite (ac.NaNO2 ) mediated nitrosative stress. METHODS AND RESULTS: Confocal microscopy and fluorescence-activated cell sorting analysis were performed to investigate the generation of reactive nitrogen and oxygen species, and redox homeostasis under nitrosative stress was also characterized. Quantitative PCR analysis revealed that the expression of ADH genes was upregulated under such condition, whereas the ACO2 gene was downregulated. Some of the enzymes of the tricarboxylic acid cycle were partially inhibited, whereas malate metabolism and alcoholic fermentation were increased under nitrosative stress. Kinetics of ethanol production was also characterized. A network analysis was conducted to validate our findings. In the presence of ac.NaNO2 , in vitro protein tyrosine nitration formation was checked by western blotting using pure alcohol dehydrogenase and aconitase. CONCLUSIONS: Alcoholic fermentation rate was increased under stress condition and this altered metabolism might be conjoined with the defence machinery to overcome the nitrosative stress. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first work of this kind where the role of metabolism under nitrosative stress has been characterized in S. cerevisiae and it will provide a base to develop an alternative method of industrial ethanol production.

Effect of Nitrogen Sources on Omega-3 Polyunsaturated Fatty Acid Biosynthesis and Gene Expression in Thraustochytriidae sp.

The molecular mechanism that contributes to nitrogen source dependent omega-3 polyunsaturated fatty acid (n-3 PUFA) synthesis in marine oleaginous protists Thraustochytriidae sp., was explored in this study. The fatty acid (FA) synthesis was significantly influenced by the supplement of various levels of sodium nitrate (SN) (1-50 mM) or urea (1-50 mM). Compared with SN (50 mM) cultivation, cells from urea (50 mM) cultivation accumulated 1.16-fold more n-3 PUFAs (49.49% docosahexaenoic acid (DHA) (w/w, of total FAs) and 5.28% docosapentaenoic acid (DPA) (w/w, of total FAs)). Strikingly higher quantities of short chain FAs (<18 carbons) (52.22-fold of that in urea cultivation) were produced from SN cultivation. Ten candidate reference genes (RGs) were screened by using four statistical methods (geNorm, NormFinder, Bestkeeper and RefFinder). MFT (Mitochondrial folate transporter) and NUC (Nucleolin) were determined as the stable RGs to normalize the RT-qPCR (real-time quantitative polymerase chain reaction) data of essential genes related to n-3 PUFAs-synthesis. Our results elucidated that the gene transcripts of delta(3,5)-delta(2,4)-dienoyl-CoA isomerase, enoyl-CoA hydratase, fatty acid elongase 3, long-chain fatty acid acyl-CoA ligase, and acetyl-CoA carboxylase were up-regulated under urea cultivation, contributing to the extension and unsaturated bond formation. These findings indicated that regulation of the specific genes through nitrogen source could greatly stimulate n-3 PUFA production in Thraustochytriidae sp.

Vasoactivity of nitrite in the iliac artery of the toad Rhinella marina.

Nitrite ([Formula: see text]) causes vasodilation in mammals due to the formation of (nitric oxide) NO by endogenous [Formula: see text] reduction in the vascular wall. In this study, we determined if a similar mechanism operates in amphibians. Dual-wire myography of the iliac artery from Rhinella marina showed that applied [Formula: see text] caused a concentration-dependent vasodilation in normoxia (21% O2; EC50: 438 µM). Hypoxia (0.63% O2) significantly increased the maximal dilation to [Formula: see text] by 5% ( P = 0.0398). The addition of oxyhemoglobin significantly increased the EC50 ( P = 0.0144; EC50: 2,236 µM) but did not affect the maximal vasodilation. In contrast, partially deoxygenated hemoglobin (90% desaturation) did not affect the EC50 ( P = 0.1189) but significantly ( P = 0.0012) increased the maximal dilation to [Formula: see text] by 11%. The soluble guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) completely abolished the response to [Formula: see text] ( P < 0.0001), and of the nitric oxide synthase inhibitors, only N5-(1-imino-3-butenyl)-l-ornithine (vinyl-l-NIO; P = 0.0028) significantly reduced the [Formula: see text] vasodilation. The xanthine oxidoreductase inhibitor allopurinol ( P = 0.927), the nitric oxide-scavenger 2-(4-carboxyphenyl)-4,5-dihydro-4,4,5,5-tetramethyl-1H-imidazolyl-1-oxy-3-oxide (C-PTIO; P = 0.478), and disruption of the endothelium ( P = 0.094) did not affect the [Formula: see text] vasodilation. Incubation of iliac arteries with 1 mM [Formula: see text] did not a cause a change in the cGMP concentration (P = 0.407). Plasma [Formula: see text] was found to be 0.86 ± 0.20 µmol/l, while nitrate ([Formula: see text]) was 19.55 ± 2.55 µmol/l. Both cygb and ngb mRNAs were expressed in the iliac artery, and it is possible that these globins facilitate [Formula: see text] reduction in hypoxia. In addition, [Formula: see text] intracellular disproportionation processes could be important in the generation of NO from [Formula: see text].

The effect of sodium nitrite infusion on renal function, brachial and central blood pressure during enzyme inhibition by allopurinol, enalapril or acetazolamide in healthy subjects: a randomized, double-blinded, placebo-controlled, crossover study.

BACKGROUND: Sodium nitrite (NaNO2) causes vasodilation, presumably by enzymatic conversion to nitric oxide (NO). Several enzymes with nitrite reducing capabilities have been discovered in vitro, but their relative importance in vivo has not been investigated. We aimed to examine the effects of NaNO2 on blood pressure, fractional sodium excretion (FENa), free water clearance (CH2O) and GFR, after pre-inhibition of xanthine oxidase, carbonic anhydrase, and angiotensin-converting enzyme. The latter as an approach to upregulate endothelial NO synthase activity. METHODS: In a double-blinded, placebo-controlled, crossover study, 16 healthy subjects were treated, in a randomized order, with placebo, allopurinol 150 mg twice daily (TD), enalapril 5 mg TD, or acetazolamide 250 mg TD. After 4 days of treatment and standardized diet, the subjects were examined at our lab. During intravenous infusion of 240 µg NaNO2/kg/hour for 2 h, we measured changes in brachial and central blood pressure (BP), plasma cyclic guanosine monophosphate (P-cGMP), plasma and urine osmolality, GFR by 51Cr-EDTA clearance, FENa and urinary excretion rate of cGMP (U-cGMP) and nitrite and nitrate (U-NOx). Subjects were supine and orally water-loaded throughout the examination day. RESULTS: Irrespective of pretreatment, we observed an increase in FENa, heart rate, U-NOx, and a decrease in CH2O and brachial systolic BP during NaNO2 infusion. P-cGMP and U-cGMP did not change during infusion. We observed a consistent trend towards a reduction in central systolic BP, which was only significant after allopurinol. CONCLUSION: This study showed a robust BP lowering, natriuretic and anti-aquaretic effect of intravenous NaNO2 regardless of preceding enzyme inhibition. None of the three enzyme inhibitors used convincingly modified the pharmacological effects of NaNO2. The steady cGMP indicates little or no conversion of nitrite to NO. Thus the effect of NaNO2 may not be mediated by NO generation. TRIAL REGISTRATION: EU Clinical Trials Register, 2013-003404-39 . Registered December 3 2013.

Effect of sodium nitrite on renal function and sodium and water excretion and brachial and central blood pressure in healthy subjects: a dose-response study.

Sodium nitrite (NaNO2) is converted to nitric oxide (NO) in vivo and has vasodilatory and natriuretic effects. Our aim was to examine the effects of NaNO2 on hemodynamics, sodium excretion, and glomerular filtration rate (GFR). In a single-blinded, placebo-controlled, crossover study, we infused placebo (0.9% NaCl) or 0.58, 1.74, or 3.48 µmol NaNO2·kg-1·h-1 for 2 h in 12 healthy subjects, after 4 days of a standard diet. Subjects were supine and water loaded. We measured brachial and central blood pressure (BP), plasma concentrations of renin, angiotensin II, aldosterone, arginine vasopressin (P-AVP), and plasma nitrite (P-[Formula: see text]), GFR by Cr-EDTA clearance, fractional excretion of sodium (FENa) free water clearance (CH2O), and urinary excretion rate of guanosine 3',5'-cyclic monophosphate (U-cGMP). The highest dose reduced brachial systolic BP (5.6 mmHg, P = 0.003), central systolic BP (5.6 mmHg, P = 0.035), and CH2O (maximum change from 3.79 to 1.27 ml/min, P = 0.031) and increased P-[Formula: see text] (from 0.065 to 0.766 µmol/l, P < 0.001), while reducing U-cGMP (from 444 to 247 pmol/min, P = 0.004). GFR, FENa, P-AVP, and the components in the renin-angiotensin-aldosterone system did not change significantly. In conclusion, intravenous NaNO2 induced a dose-dependent reduction of brachial and central BP. The hemodynamic effect was not mediated by the renin-angiotensin-aldosterone system. NaNO2 infusion resulted in a vasopressin-independent decrease in CH2O and urine output but no change in urinary sodium excretion or GFR. The lack of increase in cGMP accompanying the increase in [Formula: see text] suggests a direct effect of nitrite or nitrate on the renal tubules and vascular bed with little or no systemic conversion to NO.

Nitrite-derived nitric oxide reduces hypoxia-inducible factor 1α-mediated extracellular vesicle production by endothelial cells.

INTRODUCTION: Extracellular vesicles (EVs) are small, spherical particles enclosed by a phospholipid bilayer (∼30-1000 nm) released from multiple cell types, and have been shown to have pathophysiological roles in a plethora of disease states. The transcription factor hypoxia-inducible factor-1 (HIF-1) allows for adaptation of cellular physiology in hypoxia and may permit the enhanced release of EVs under such conditions. Nitric oxide (NO) plays a pivotal role in vascular homeostasis, and can modulate the cellular response to hypoxia by preventing HIF-1 accumulation. We aimed to selectively target HIF-1 via sodium nitrite (NaNO2) addition, and examine the effect on endothelial EV, size, concentration and function, and delineate the role of HIF-1 in EV biogenesis. METHODS: Endothelial (HECV) cells were exposed to hypoxic conditions (1% O2, 24 h) and compared to endothelial cells exposed to normoxia (21% O2) with and without the presence of sodium nitrite (NaNO2) (30 µM). Allopurinol (100 µM), an inhibitor of xanthine oxidoreductase, was added both alone and in combination with NaNO2 to cells exposed to hypoxia. EV and cell preparations were quantified by nanoparticle tracking analysis and confirmed by electron microscopy. Western blotting and siRNA were used to confirm the role of HIF-1α and HIF-2α in EV biogenesis. Flow cytometry and time-resolved fluorescence were used to assess the surface and intravesicular protein content. RESULTS: Endothelial (HECV) cells exposed to hypoxia (1% O2) produced higher levels of EVs compared to cells exposed to normoxia. This increase was confirmed using the hypoxia-mimetic agent desferrioxamine. Treatment of cells with sodium nitrite (NaNO2) reduced the hypoxic enhancement of EV production. Treatment of cells with the xanthine oxidoreductase inhibitor allopurinol, in addition to NaNO2 attenuated the NaNO2-attributed suppression of hypoxia-mediated EV release. Transfection of cells with HIF-1α siRNA, but not HIF-2α siRNA, prior to hypoxic exposure prevented the enhancement of EV release. CONCLUSION: These data provide evidence that hypoxia enhances the release of EVs in endothelial cells, and that this is mediated by HIF-1α, but not HIF-2α. Furthermore, the reduction of NO2- to NO via xanthine oxidoreductase during hypoxia appears to inhibit HIF-1α-mediated EV production.

Volatile organic compounds released from Microcystis flos-aquae under nitrogen sources and their toxic effects on Chlorella vulgaris.

Eutrophication promotes massive growth of cyanobacteria and algal blooms, which can poison other algae and reduce biodiversity. To investigate the differences in multiple nitrogen (N) sources in eutrophicated water on the emission of volatile organic compounds (VOCs) from cyanobacteria, and their toxic effects on other algal growth, we analyzed VOCs emitted from Microcystis flos-aquae with different types and concentrations of nitrogen, and determined the effects under Normal-N and Non-N conditions on Chlorella vulgaris. M. flos-aquae released 27, 22, 20, 27, 19, 25 and 17 compounds, respectively, with NaNO3, NaNO2, NH4Cl, urea, Ser, Lys and Arg as the sole N source. With the reduction in N amount, the emission of VOCs was increased markedly, and the most VOCs were found under Non-N condition. C. vulgaris cell propagation, photosynthetic pigment and Fv/Fm declined significantly following exposure to M. flos-aquae VOCs under Non-N condition, but not under Normal-N condition. When C. vulgaris cells were treated with two terpenoids, eucalyptol and limonene, the inhibitory effects were enhanced with increasing concentrations. Therefore, multiple N sources in eutrophicated water induce different VOC emissions from cyanobacteria, and reduction in N can cause nutrient competition, which can result in emissions of more VOCs. Those VOCs released from M. flos-aquae cells under Non-N for nutrient competition can inhibit other algal growth. Among those VOCs, eucalyptol and limonene are the major toxic agents.

Correlations between the Electronic Properties of Shewanella oneidensis Cytochrome c Nitrite Reductase (ccNiR) and Its Structure: Effects of Heme Oxidation State and Active Site Ligation.

The electrochemical properties of Shewanella oneidensis cytochrome c nitrite reductase (ccNiR), a homodimer that contains five hemes per protomer, were investigated by UV-visible and electron paramagnetic resonance (EPR) spectropotentiometries. Global analysis of the UV-vis spectropotentiometric results yielded highly reproducible values for the heme midpoint potentials. These midpoint potential values were then assigned to specific hemes in each protomer (as defined in previous X-ray diffraction studies) by comparing the EPR and UV-vis spectropotentiometric results, taking advantage of the high sensitivity of EPR spectra to the structural microenvironment of paramagnetic centers. Addition of the strong-field ligand cyanide led to a 70 mV positive shift of the active site's midpoint potential, as the cyanide bound to the initially five-coordinate high-spin heme and triggered a high-spin to low-spin transition. With cyanide present, three of the remaining hemes gave rise to distinctive and readily assignable EPR spectral changes upon reduction, while a fourth was EPR-silent. At high applied potentials, interpretation of the EPR spectra in the absence of cyanide was complicated by a magnetic interaction that appears to involve three of five hemes in each protomer. At lower applied potentials, the spectra recorded in the presence and absence of cyanide were similar, which aided global assignment of the signals. The midpoint potential of the EPR-silent heme could be assigned by default, but the assignment was also confirmed by UV-vis spectropotentiometric analysis of the H268M mutant of ccNiR, in which one of the EPR-silent heme's histidine axial ligands was replaced with a methionine.

Short communication: Use of a mixture of sodium nitrite, sodium benzoate, and potassium sorbate in aerobically challenged silages.

Aerobic instability is still a common problem with many types of silages, particularly well-fermented silages. This study evaluated the effect of adding an additive mixture based on sodium nitrite, sodium benzoate, and potassium sorbate to a variety of crop materials on fermentation quality and aerobic stability of silages. Ensiling conditions were challenged by using a low packing density (104±4.3kg of dry matter/m(3)) of forage and allowing air ingression into silos (at 14 and 7 d before the end of the storage, for 8 h per event). Additive-treated silages were found to have significantly lower pH and reduced formation of ammonia-N, 2.3-butanediol, and ethanol compared with untreated control silages. Yeast growth was significantly reduced by additive treatment in comparison with untreated control silage. Consequently, additive-treated silages were considerably more aerobically stable (6.7 d) than untreated control silages (0.5 d). Overall, adding 5mL/kg of fresh crop of the additive based on sodium nitrite, sodium benzoate, and potassium sorbate reduced undesirable microorganisms in silages and thereby provided suitable ensiling conditions and prolonged aerobic stability, even under air-challenged laboratory ensiling conditions.

Optimization of inorganic carbon sources to improve the carbon fixation efficiency of the non-photosynthetic microbial community with different electron donors.

As the non-photosynthetic microbial community (NPMC) isolated from seawaters utilized inorganic carbon sources for carbon fixation, the concentrations and ratios of Na2CO3, NaHCO3, and CO2 were optimized by response surface methodology design. With H2 as the electron donor, the optimal carbon sources were 270 mg/L Na2CO3, 580 mg/L NaHCO3, and 120 mg/L CO2. The carbon fixation efficiency in response to total organic carbon (TOC) was up to 30.59 mg/L with optimal carbon sources, which was about 50% higher than that obtained with CO2 as the sole carbon source. The mixture of inorganic carbon sources developed a buffer system to prevent acidification or alkalization of the medium caused by CO2 or Na2CO3, respectively. Furthermore, CO2 and HCO3(-), the starting points of carbon fixation in the pathways of Calvin-Benson-Bassham and 3-hydroxypropionate cycles, were provided by the carbon source structure to facilitate carbon fixation by NPMC. However, in the presence of mixed electron donors composed of 1.25% Na2S, 0.50% Na2S2O3, and 0.457% NaNO2, the carbon source structure did not exhibit significant improvement in the carbon fixation efficiency, when compared with that achieved with CO2 as the sole carbon source. The positive effect of mixed electron donors on inorganic carbon fixation was much higher than that of the carbon source structure. Nevertheless, the carbon source structure could be used as an alternative to CO2 when using NPMC to fix carbon in industrial processes.

Sodium nitrite in patients with peripheral artery disease and diabetes mellitus: safety, walking distance and endothelial function.

Nitrite stores decrease after exercise in patients with peripheral artery disease (PAD) and diabetes represents decreased nitric oxide (NO) bioavailability that may contribute to endothelial dysfunction and limit exercise duration. The primary objective of this placebo-controlled study was the safety and tolerability of multiple doses of oral sodium nitrite in patients with PAD, predominantly with diabetes, over a period of 10 weeks. The primary efficacy endpoint was endothelial flow-mediated dilatation (FMD) and secondary efficacy endpoints included a 6-minute walk test and quality of life assessment. Of the 55 subjects, the most common side effects attributed to sodium nitrite were a composite of headache and dizziness occurring in 21% with the 40 mg dose and 44% with the 80 mg dose. There was no clinically significant elevation of methemoglobin. FMD non-significantly worsened in the placebo and 40 mg groups, but was stable in the 80 mg group. Diabetic patients receiving 80 mg had significantly higher FMD compared with the placebo and 40 mg groups. There was no significant change in 6-minute walk test or quality of life parameters over time compared to placebo. In conclusion, sodium nitrite therapy is well tolerated in patients with PAD. The possible clinical benefit of sodium nitrite should be studied in a larger and fully powered trial.

Ameliorative effect of Odontonema cuspidatum extract against testicular damage induced by sodium nitrite in rats.

Background: Sodium nitrite (NaNO2) is a chemical substance used to enhance taste, add color, and keep food products fit for consumption for a longer time. NaNO2 gives rise to a negative adverse effect on male reproductive function. Odontonema cuspidatum (OC) is a natural plant that possesses antioxidant capacity. Aim: Our research evaluates the potential beneficial effect of OC extract on the harmful effects caused by NaNO2 on the testicular tissue and sperm characteristics of male rats. Methods: Four groups with a total of forty rats: the control, the NaNO2-received group, the OC-administered group, and the fourth group received both NaNO2 and OC. All groups were administered daily for two months. Sperm characteristics, testicular antioxidant status, qRT-PCR, and histopathological changes were evaluated. Results: Coadministration of NaNO2 and OC, in comparison with NaNO2 alone, contributed to a notable enhancement in acrosomal integrity, decreasing sperm abnormalities and restoring serum testosterone levels. Moreover, such coadministration reduced the oxidative stress marker, malondialdehyde (MDA), and increased superoxide dismutase (SOD) in testicular tissue, lowering TNF-α gene expression, and increasing the expression of P450scc and StAR genes. In addition, the NaNO2 and OC combination decreased the testicular histopathological changes and the Caspase-3 and Proliferating cell nuclear antigen (PCNA) immunoexpression in seminiferous tubules compared with the NaNO2 group. Conclusion: The extract of OC exhibited the ability to decrease oxidative stress and ameliorate the detrimental effects caused by NaNO2.

Pennisetum glaucum Protein Extract Protects RBC, Liver, Kidney, Small Intestine from Oxidative Damage and Exhibits Anticoagulant, Antiplatelet Activity.

High level of exogenous ROS in the circulation affects RBC membrane integrity which facilitates the generation of endogenous RBC ROS, implicated in series of physiological changes primarily associated with thrombosis and vital tissue damage. Although, Pennisetum glaucum (pearl millet) stores abundance of proteins, their therapeutic potential is least explored. Thus, the purpose of this study is to examine the role of Pennisetum Glaucum Protein Extract (PGE) on oxidative stress induced cell/tissue damage and thrombosis.In this investigation, protein characterization was done by using SDS-PAGE, Native-PAGE, PAS-staining and HPLC. In-vitro oxidative stress was induced in RBC using sodium nitrite. While, in-vivo oxidative stress was induced in experimental rats using diclofenac. Stress markers and biochemical parameters were evaluated. Role of PGE on thrombosis was assessed by using, in-vitro plasma recalcification time, activated partial thromboplastin time, prothrombin time, mouse tail bleeding time (In-vivo) and platelet aggregation.PGE revealed varied range of molecular weight proteins on SDS-PAGE. PGE normalized the sodium nitrite induced oxidative damage of RBC and diclofenac induced oxidative damage in liver, kidney and small intestine. PGE exhibited anticoagulant effect by increasing the coagulation time of both PRP and PPP and mouse tail bleeding time. Furthermore, PGE prolonged the clotting time of only APTT but did not affect PT. PGE inhibited agonists ADP and epinephrine induced platelet aggregation.Our findings suggest, PGE could be a better contender in the management of oxidative stress and its associated diseases. ABBREVIATIONS: PGEPennisetum Glaucum protein ExtractAPPTActivated Partial Thromboplastin TimePTProthrombin TimeROSReactive Oxygen SpeciesPRPPlatelet Rich PlasmaPPPPlatelet Poor PlasmaSDS-PAGESodium Dodecyl Sulfate-Polyacrylamide Gel ElectrophoresisPASPeriodic Acid-schiff StainingODOptical DensityINRInternational Normalized RatioPBSPhosphate Buffered SalineSODSuperoxide DismutaseTCATrichloro Acetatic AcidDTNBDi-Thio-bis-NitroBenzoic acidSGOTSerum Glutamate Oxaloacetate TransaminaseSGPTSerum Glutamate Pyruvate TransaminaseALPAlkaline PhosphataseDFCDiclofenacSylSilymarinMEDMinimum Edema DoseMHDMinimum Hemorrhagic Dose.

Inactivation of the potent Pseudomonas aeruginosa cytotoxin pyocyanin by airway peroxidases and nitrite.

Pyocyanin (1-hydroxy-N-methylphenazine, PCN) is a cytotoxic pigment and virulence factor secreted by the human bacterial pathogen, Pseudomonas aeruginosa. Here, we report that exposure of PCN to airway peroxidases, hydrogen peroxide (H(2)O(2)), and NaNO(2) generates unique mononitrated PCN metabolites (N-PCN) as revealed by HPLC/mass spectrometry analyses. N-PCN, in contrast to PCN, was devoid of antibiotic activity and failed to kill Escherichia coli and Staphylococcus aureus. Furthermore, in contrast to PCN, intratracheal instillation of N-PCN into murine lungs failed to induce a significant inflammatory response. Surprisingly, at a pH of ∼7, N-PCN was more reactive than PCN with respect to NADH oxidation but resulted in a similar magnitude of superoxide production as detected by electron paramagnetic resonance and spin trapping experiments. When incubated with Escherichia coli or lung A549 cells, PCN and N-PCN both led to superoxide formation, but lesser amounts were detected with N-PCN. Our results demonstrate that PCN that has been nitrated by peroxidase/H(2)O(2)/NO(2)(-) systems possesses less cytotoxic/proinflammatory activity than native PCN. Yield of N-PCN was decreased by the presence of the competing physiological peroxidase substrates (thiocyonate) SCN(-) (myeloperoxidase, MPO, and lactoperoxidase, LPO) and Cl(-) (MPO), which with Cl(-) yielded chlorinated PCNs. These reaction products also showed decreased proinflammatory ability when instilled into the lungs of mice. These observations add important insights into the complexity of the pathogenesis of lung injury associated with Pseudomonas aeruginosa infections and provide additional rationale for exploring the efficacy of NO(2)(-) in the therapy of chronic Pseudomonas aeruginosa airway infection in cystic fibrosis.

Induction of colonic aberrant crypts in mice by feeding apparent N-nitroso compounds derived from hot dogs.

Nitrite-preserved meats (e.g., hot dogs) may help cause colon cancer because they contain N-nitroso compounds. We tested whether purified hot-dog-derived total apparent N-nitroso compounds (ANC) could induce colonic aberrant crypts, which are putative precursors of colon cancer. We purified ANC precursors in hot dogs and nitrosated them to produce ANC. In preliminary tests, CF1 mice received 1 or 3 i.p. injections of 5 mg azoxymethane (AOM)/kg. In Experiments 1 and 2, female A/J mice received ANC in diet. In Experiment 1, ANC dose initially dropped sharply because the ANC precursors had mostly decomposed but, later in Experiment 1 and throughout Experiment 2, ANC remained at 85 nmol/g diet. Mice were killed after 8 (AOM tests) or 17-34 (ANC tests) wk. Median numbers of aberrant crypts in the distal 2 cm of the colon for 1 and 3 AOM injections, CF1 controls, ANC (Experiment 1), ANC (Experiment 2),and untreated A/J mice were 31, 74, 12, 20, 12, and 5-6, with P < 0.01 for both ANC tests. Experiment 2 showed somewhat increased numbers of colonic mucin-depleted foci in the ANC-treated group. We conclude that hot-dog-derived ANC induced significant numbers of aberrant crypts in the mouse colon.

[Study on eliminating sodium nitrite and blocking nitrosamine synthesis by anthocyanin from skin of Alpinia galanga].

This study was performed to determine the ability of eliminating sodium nitrite and blocking nitrosamine synthesis by anthocyanin from the skin of Alpinia galanga. purified by macroporous resin. The test was conducted under the condition of the simulated human gastric juice (pH 3.0, 37 degrees C) with VitC as positive control. The results showed that the max capability of eliminating sodium nitrite was 87.14%, which is 1.6 times sronger than that of VitC, and the max capability of blocking nitrosamine synthesis was 97.82%, which is 8 times sronger than that of VitC.

Protective Effect of Selenium on the Oxidative Damage of Kidney Cells Induced by Sodium Nitrite in Grass Carp (Ctenopharyngodon idellus).

The present study was conducted to investigate the protective effects of selenium on the oxidative damage of kidney cells (CIK) caused by nitrite exposure in grass carp (Ctenopharyngodon idella). Cells were pre-incubated by Na2SeO3 (10 µmol/L) for 12 h and then exposed to NaNO2 (25 mg/L) for 24 h, the cell viability, apoptosis, gene expression, and antioxidant enzyme activity were assayed. The results show that nitrite reduced cell viability and induced apoptosis, and the activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) as well as the sod, cat, and gpx genes reduced (p < 0.05), while the intracellular calcium ion concentration increased (p < 0.05). Interestingly, selenium treatment significantly alleviated the nitrite induced changes in cell growth, apoptosis, and calcium influx. The cell viability after low-concentration selenium treatment is higher than that of normal cells (p < 0.05). CIK cells were pre-incubated with Na2SeO3 and then exposed to NaNO2, the antioxidant indicators could be maintained at normal levels. And compared with nitrite exposure, intracellular calcium ion concentration and apoptotic rate of selenium-incubated still decreased. The expressions of Nrf2 and Keap1 genes increased significantly in CIK cells treated with sodium selenite for 12 h, and the same trend as the enzyme activities of this group. The results show that the supplement of selenium can enhance the cell's resistance to sodium nitrite exposure to a certain extent, by alleviating the antioxidant imbalance, high apoptosis rate, and intracellular calcium ion disturbance caused by nitrite exposure. And the Nrf2-Keap1 pathway may play an important role in the process.

Impact of Thymus vulgaris extract on sodium nitrite-induced alteration of renal redox and oxidative stress: Biochemical, molecular, and immunohistochemical study.

Thyme (Thymus vulgaris) is an herbal plant with pleiotropic medicinal properties. In this study, we examined the possible protective effect of an ethanolic extract of thyme leaves against the renal oxidative stress induced by sodium nitrite (NaNO2 ). Male Swiss mice received either saline or thyme extract for 15 days (0.5 g/kg body weight, orally). NaNO2 (60 mg/kg) was injected intraperitoneally at Day 14. The protective group received the thyme extract for 15 days and NaNO2 on Day 14. Blood and kidney samples were taken from all groups to measure serum urea, blood urea nitrogen (BUN), creatinine, serum, tissue antioxidant activity, and the inflammatory cytokines IL-1ß and IL-6. Quantitative real-time PCR (qRT-PCR) was used to examine the expression of kidney injury marker-1 (Kim-1), TNF-α, nuclear factor erythroid-2 related factor 2 (Nrf2), and hemoxygenase-1 (HO-1), all of which are associated with kidney redox and oxidative stress. Pretreatment with thyme extract reduced the effects of NaNO2 on urea, BUN, and creatinine, and reversed its effect on tissue and serum antioxidants. NaNO2 -induced nephritis as demonstrated by the upregulation in mRNA expression of Kim-1 and TNF-α, which was, however, recovered and protected by pretreatment with thyme extract. Expression of Nrf2 and HO-1 was upregulated by treatment with thyme extract and downregulated by NaNO2 intoxication. NaNO2 -induced congestion in glomeruli and dilatation of the renal tubules, conditions that were restored in the group pretreated with thyme extract. NaNO2 upregulated Bax immunoreactivity and caused apoptosis in renal structures. Thus, thyme extract is effective in managing the renal toxicity associated with oxidative stress and renal redox. PRACTICAL APPLICATIONS: The results from this study have shown that use of thyme extract may promote better health due to its high antioxidant activity. For instance, it could be ingested to alleviate the symptoms of renal inflammation and oxidative stress associated with nitrite toxicity. Thyme extract regulated renal redox, oxidative stress, antioxidant levels, and inflammation-associated genes at the molecular, biochemical, and cellular immunohistochemical levels.

Plant and cyanobacterial hemoglobins reduce nitrite to nitric oxide under anoxic conditions.

The ability of ferrous hemoglobins to reduce nitrite to form nitric oxide has been demonstrated for hemoglobins from animals, including myoglobin, blood cell hemoglobin, neuroglobin, and cytoglobin. In all cases, the rate constants for the bimolecular reactions with nitrite are relatively slow, with maximal values of ~5 M(-1) s(-1) at pH 7. Combined with the relatively low concentrations of nitrite found in animal blood plasma (normally no greater than 13 µM), these slow reaction rates are unlikely to contribute significantly to hemoglobin oxidation, nitrite reduction, or NO production. Plants and cyanobacteria, however, must contend with much higher (millimolar) nitrite concentrations necessitated by assimilatory nitrogen metabolism during hypoxic growth, such as the conditions commonly found during flooding or in waterlogged soil. Here we report rate constants for nitrite reduction by a ferrous plant hemoglobin (rice nonsymbiotic hemoglobin 1) and a ferrous cyanobacterial hemoglobin from Synechocystis that are more than 10 times faster than those observed for animal hemoglobins. These rate constants, along with the relatively high concentrations of nitrite present during hypoxia, suggest that plant and cyanobacterial hemoglobins could serve as anaerobic nitrite reductases in vivo.