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.

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.

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.

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.

[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.

The effect of nitric-oxide-related supplements on human performance.

Nitric oxide (NO) has led a revolution in physiology and pharmacology research during the last two decades. This labile molecule plays an important role in many functions in the body regulating vasodilatation, blood flow, mitochondrial respiration and platelet function. Currently, it is known that NO synthesis occurs via at least two physiological pathways: NO synthase (NOS) dependent and NOS independent. In the former, L-arginine is the main precursor. It is widely recognized that this amino acid is oxidized to NO by the action of the NOS enzymes. Additionally, L-citrulline has been indicated to be a secondary NO donor in the NOS-dependent pathway, since it can be converted to L-arginine. Nitrate and nitrite are the main substrates to produce NO via the NOS-independent pathway. These anions can be reduced in vivo to NO and other bioactive nitrogen oxides. Other molecules, such as the dietary supplement glycine propionyl-L-carnitine (GPLC), have also been suggested to increase levels of NO, although the physiological mechanisms remain to be elucidated. The interest in all these molecules has increased in many fields of research. In relation with exercise physiology, it has been suggested that an increase in NO production may enhance oxygen and nutrient delivery to active muscles, thus improving tolerance to physical exercise and recovery mechanisms. Several studies using NO donors have assessed this hypothesis in a healthy, trained population. However, the conclusions from these studies showed several discrepancies. While some reported that dietary supplementation with NO donors induced benefits in exercise performance, others did not find any positive effect. In this regard, training status of the subjects seems to be an important factor linked to the ergogenic effect of NO supplementation. Studies involving untrained or moderately trained healthy subjects showed that NO donors could improve tolerance to aerobic and anaerobic exercise. However, when highly trained subjects were supplemented, no positive effect on performance was indicated. In addition, all this evidence is mainly based on a young male population. Further research in elderly and female subjects is needed to determine whether NO supplements can induce benefit in exercise capacity when the NO metabolism is impaired by age and/or estrogen status.

Effect of rosemary extract dose on lipid oxidation, colour stability and antioxidant concentrations, in reduced nitrite liver pâtés.

The oxidative stability of liver pâté was investigated in relation to different doses of rosemary extract (RE) and sodium nitrite. Colour stability, lipid oxidation (TBARS) and concentrations of ascorbic acid, α-tocopherol, carnosic acid and nitrite were measured on the batters before cooking and on the cooked liver pâté before and after exposure to light and air for 48 h at 4°C. Results showed that the use of RE significantly reduced lipid oxidation, whereas it had no effect on colour stability. Ascorbic acid and nitrite concentrations were significantly higher and lower respectively when RE was added. RE dose-dependently increased the concentration of carnosic acid. Lower sodium nitrite doses resulted in significantly lower nitrite concentrations and slightly lower TBARS values. It was concluded that in liver pâté sodium nitrite levels may be lowered to 80 mg/kg without negatively affecting colour and lipid stability and that the use of RE may help in maintaining lipid stability.

Characterization of nitrated phenolic compounds for their anti-oxidant, pro-oxidant, and nitration activities.

Coffee is one of the most widely consumed beverages worldwide. Evidence of the health benefits and the important contribution of coffee brew to the intake of anti-oxidants in the diet has increased coffee consumption. Chlorogenic acid (ChA) and caffeic acid (CaA) are the major phenolic compounds in coffee. However, phenolic compounds, which are generally effective anti-oxidants, can become pro-oxidants in the presence of Cu(2+) to induce DNA damage under certain conditions. On the other hand, sodium nitrite (NaNO(2)) is widely used as a food additive to preserve and tinge color on cured meat and fish. It is possible that phenolic compounds react with NaNO(2) under acidic conditions, such as gastric juice. In this study, we identified compounds produced by the reaction between ChA or CaA in coffee and NaNO(2) in artificial gastric juice. The identified phenolic compounds and nitrated phenolic compounds were assessed for their anti-oxidant, pro-oxidant, and nitration activities by performing an in vitro assay. The nitrated phenolic compounds seemed to show increased anti-oxidant activity and decreased pro-oxidant activity. However, one nitrated CaA compound that has a furoxan ring showed the ability to release NO(2)(-) in the neutral condition.

Nitrite supplementation reverses vascular endothelial dysfunction and large elastic artery stiffness with aging.

We tested the hypothesis that short-term nitrite therapy reverses vascular endothelial dysfunction and large elastic artery stiffening with aging, and reduces arterial oxidative stress and inflammation. Nitrite concentrations were lower (P < 0.05) in arteries, heart, and plasma of old (26-28 month) male C57BL6 control mice, and 3 weeks of sodium nitrite (50 mg L(-1) in drinking water) restored nitrite levels to or above young (4-6 month) controls. Isolated carotid arteries of old control mice had lower acetylcholine (ACh)-induced endothelium-dependent dilation (EDD) (71.7 ± 6.1% vs. 93.0 ± 2.0%) mediated by reduced nitric oxide (NO) bioavailability (P < 0.05 vs. young), and sodium nitrite restored EDD (95.5 ± 1.6%) by increasing NO bioavailability. 4-Hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl (TEMPOL), a superoxide dismutase (SOD) mimetic, apocynin, a nicotinamide adenine dinucleotide phosphate-oxidase (NADPH) inhibitor, and sepiapterin (exogenous tetrahydrobiopterin) each restored EDD to ACh in old control, but had no effect in old nitrite-supplemented mice. Old control mice had increased aortic pulse wave velocity (478 ± 16 vs. 332 ± 12 AU, P < 0.05 vs. young), which nitrite supplementation lowered (384 ± 27 AU). Nitrotyrosine, superoxide production, and expression of NADPH oxidase were ∼100-300% greater and SOD activity was ∼50% lower in old control mice (all P < 0.05 vs. young), but were ameliorated by sodium nitrite treatment. Inflammatory cytokines were markedly increased in old control mice (P < 0.05), but reduced to levels of young controls with nitrite supplementation. Short-term nitrite therapy reverses age-associated vascular endothelial dysfunction, large elastic artery stiffness, oxidative stress, and inflammation. Sodium nitrite may be a novel therapy for treating arterial aging in humans.

Prooxidant effects of the combination of green tea extract and sodium nitrite for accelerating lipolysis and lipid oxidation in pepperoni during storage.

UNLABELLED: The individual and interactive effects of green tea (GT) extract and sodium nitrite (NT) on lipolysis and lipid oxidation were investigated in pepperoni during processing and storage (4 °C). Pepperoni was treated with GT at concentrations of 0.02%, 0.05%, 0.1%, 0.2%, 0.5%; and NT from 0.003% to 0.015% in increments of 0.003%; and a combination of 0.05% GT and 0.009% NT (GT/NT). The pH, color, residual nitrite, fatty acid (FA), and 2-thiobarbituric acid reactive substances (TBARS) values were measured. The results showed inhibitory effects on TBARS value were concentration dependent. The largest effect was at 0.05% for GT and 0.009% for NT. Adding 0.05% GT significantly decreased (P < 0.05) the TBARS values but did not affect color values during storage. NT (0.009%) was more effective than GT (0.05%) for maintaining lower TBARS values (P < 0.05). Total fatty acids were not affected by 0.05% GT and 0.009% NT treatments. However, adding GT/NT showed a significant prooxidant effect (P < 0.05) in pepperoni, which accelerated lipolysis and lipid oxidation significantly (P < 0.05) during storage, with depletion of nitrite. This suggests that adding GT and an appropriate amount of NT into pepperoni could promote lipolysis and lipid oxidation during storage, as compared to NT alone. PRACTICAL APPLICATION: When using GT extract as an antioxidant in industrial production of pepperoni, the interaction effect with NT should be considered, as it may lead to a prooxidant effect during refrigerated storage.

The depletion of sodium nitrite by lactic acid bacteria isolated from kimchi.

Nitrites, whether added or naturally occurring in foods, are potential carcinogens, and controlling their concentrations is important for maintaining a safe food supply. In this study we investigated the depletion of sodium nitrite (150 microg/mL) during the fermentation in Lactobacilli MRS broth at 5, 10, 15, 20, 25, 30, and 36 degrees C by lactic acid bacteria (LAB-A, -B, -C, and -D) isolated from kimchi and Leuconostoc mesenteroides strain KCTC3100. The four species of lactic acid bacteria isolated from kimchi were identified as L. mesenteroides, and all produced depletion of less than 20% of sodium nitrite after 10 days of incubation at 5 degrees C. There was less than 40% depletion after 9 days at 10 degrees C, 86.4-92.8% after 7 days at 15 degrees C, 81.4-87.8% after 4 days and more than 90.0% after 5 days at 20 degrees C, 76.3-85.7% after 3 days and more than 90.0% after 5 days at 25 degrees C, and more than 90.0% after 2 days at 30 and 36 degrees C. The depletion by LAB isolates was similar or higher than that by L. mesenteroides strain KCTC3100, and in particular, the LAB-D strain showed the highest depletion effect of all the strains tested, up to 15 degrees C. From these results, the strains isolated from kimchi were very effective for the depletion of sodium nitrite at high temperature, and all sodium nitrite was depleted at the initial period of incubation (1-2 days) at 30 and 36 degrees C. But as the temperature was lowered, the depletion effect of sodium nitrite was decreased in all the strains tested from kimchi. This illustrates that the depletion of nitrite by each strain is subject to the influence of temperatures.

In vitro evaluation of a new treatment for urinary tract infections caused by nitrate-reducing bacteria.

Dietary and endogenous nitrates are excreted in urine, and during infection with nitrate-reducing bacteria they are reduced to nitrite. At a low pH nitrite is converted to a variety of nitrogen oxides that are toxic to bacteria. We hypothesized that acidification of nitrite-rich infected urine would result in the killing of the nitrate-reducing bacteria. An Escherichia coli control strain and a mutant lacking nitrate reductase activity were preincubated in urine supplemented with sodium nitrate (0 to 10 mM) at pH 7.0. Then, the nitrite-containing bacterial culture was transferred (and diluted 1/10) to slightly acidic urine (pH 5 and 5.5) containing ascorbic acid (10 mM) and growth was monitored. The control strain produced nitrite in amounts related to the amount of nitrate added. This strain was killed when the culture was transferred to acidic urine. In contrast, the mutant that did not produce nitrite retained full viability. When control bacteria were grown in acidic urine with nitrate and ascorbic acid present from the start of the experiment, no inhibition of growth was noted. The MICs and minimal bactericidal concentrations of sodium nitrite-ascorbic acid in acidic urine were comparable to those of conventional antibiotics. Preincubation of nitrate-reducing E. coli in nitrate-rich urine leads to the accumulation of nitrite. Subsequent acidification of the urine results in generation of nitrogen oxides that are bactericidal. Killing, however, requires a sequential procedure in which the bacteria are first allowed to grow in a nitrate-rich neutral environment, later followed by acidification. We speculate that ingestion of nitrate followed some hours later by acidification of urine could be a new therapeutic strategy for the treatment of urinary tract infections.

Inactivation of myocardial dihydrolipoamide dehydrogenase by myeloperoxidase systems: effect of halides, nitrite and thiol compounds.

Dihydrolipoamide dehydrogenase (LADH) lipoamide reductase activity decreased whereas enzyme diaphorase activity increased after LADH treatment with myeloperoxidase (MPO) dependent systems (MPO/H2O2/halide, MPO/NADH/halide and MPO/H2O2/nitrite systems. LADH inactivation was a function of the composition of the inactivating system and the incubation time. Chloride, iodide, bromide, and the thiocyanate anions were effective complements of the MPO/H2O2 system. NaOCl inactivated LADH, thus supporting hypochlorous acid (HOCl) as putative agent of the MPO/H2O2/NaCl system. NaOCl and the MPO/H2O2/NaCl system oxidized LADH thiols and NaOCl also oxidized LADH methionine and tyrosine residues. LADH inactivation by the MPO/NADH/halide systems was prevented by catalase and enhanced by superoxide dismutase, in close agreement with H2O2 production by the LADH/NADH system. Similar effects were obtained with lactoperoxidase and horse-radish peroxidase supplemented systems. L-cysteine, N-acetylcysteine, penicillamine, N-(2-mercaptopropionylglycine), Captopril and taurine protected LADH against MPO systems and NaOCl. The effect of the MPO/H2O2/NaNO2 system was prevented by MPO inhibitors (sodium azide, isoniazid, salicylhydroxamic acid) and also by L-cysteine, L-methionine, L-tryptophan, L-tyrosine, L-histidine and reduced glutathione. The summarized observations support the hypothesis that peroxidase-generated "reactive species" oxidize essential thiol groups at LADH catalytic site.

Forward mutation of S. typhimurium by smokeless tobacco extracts.

Mutagenicity of 4 popular brands of smokeless tobaccos was studied using a S. typhimurium forward mutation assay. Aqueous extracts of 4 brands and dichloromethane and methanol extracts of 1 of the 4 brands of smokeless tobacco's did not induce significant mutagenicity either in the presence or absence of metabolic activation. Aqueous and organic extracts were however mutagenic when treated with physiological levels of sodium nitrite (0.25 mM) at acidic pH and without metabolic activation. The results indicate that smokeless tobacco contain polar and non-polar chemicals which become mutagenic to S. typhimurium under nitrosation conditions.

The intragastric host-mediated assay for the assessment of the formation of direct mutagens in vivo.

The intragastric host-mediated assay (h.m.a.) was devised and carried out with a view to assessing the formation of direct mutagens in the gastrointestinal tract of mammals. The h.m.a. consists in the injection of nitrosable compounds, NaNO2 and cells of the yeast S. pombe, by gavage into the animals' stomachs and in the recovery of the target cells from the faeces for mutation-induction analysis. Methylurea was chosen as a model nitrosable compound, and the effects of nitrosation modulators such as ascorbic acid and thiocyanate were studied. Cimetidine, a drug nitrosable in vitro, was tested with the system. Positive results were obtained only at very large doses and in artificially produced low pH. The new host-mediated assay seems to be efficient in revealing the formation, in vivo, of direct, short-living mutagens.

Inhibitory effects of phenolics, teas and saliva on the formation of mutagenic nitrosation products of salted fish.

The objectives of this study were to simulate in vitro some of the conditions that may prevail in man during the ingestion of a meal and to quantitate the inhibitory effect of phenolics and phenolic-containing beverages on the formation of mutagenic nitrosation products. The test system consisted of nitrosating (pH 2, 1 h, 37 degrees C) an aqueous fraction of a salt-preserved Chinese fish (Pak Wik) with or without the inhibitors to be tested and estimating the frequency of his+ revertants per survivor of Salmonella typhimurium (strain TA1535). The phenolics and teas were added to the nitrosation mixture. Catechin, chlorogenic acid, gallic acid, pyrogallol and tannic acid suppressed the formation of mutagenic nitrosation products. The inhibitory efficiency was comparable to that of ascorbic acid. A Japanese, a Chinese and a Ceylonese tea also prevented the formation of mutagenic nitrosated fish products at doses which are usually consumed by man. Moreover, saliva exerted an inhibitory effect. The inhibitory effect was not additive when the phenolics or saliva were added concurrently to the nitrosation mixture. The possibility that phenolics are involved in the apparent chemopreventive effect of fruits and vegetables is discussed.