Characterization and genomic analysis of the lytic bacteriophage vB_EclM_HK6 as a potential approach to biocontrol the spread of Enterobacter cloacae contaminating food.

BACKGROUND: Increased prevalence of Enterobacter cloacae within food products underscores food as an underexplored reservoir for antibiotic resistance, thus requiring particular intervention. Bacteriophages have been explored as a promising approach for controlling bacterial growth in different matrices. Moreover, their specific interaction and self-replication, put them apart from traditional methods for controlling bacteria in different matrices. METHODS: Sixteen Enterobacter cloacae strains were recovered from raw chicken. These strains were used to isolate bacteriophages using enrichment protocol. The broad-spectrum bacteriophage was evaluated in terms of thermal, pH, shearing stress and storge. Moreover, its infection kinetics, in vitro antibacterial activity, cytotoxicity were also assessed. Genomic sequencing was performed to exclude any potential virulence or resistance genes. Finally, the capability of the isolated phages to control bacterial growth in different chicken samples was assessed alone and in combination with sodium nitrite. RESULTS: The lytic bacteriophage vB_EclM_HK6 was isolated and showed the broadest spectrum being able to infect 8/16 E. cloacae strains with a lytic activity against its host strain, E. cloacae EC21, as low as MOI of 10-6. The phage displays a latent period of 10 min and burst size of 115 ± 44 and resistance frequency of 5.7 × 10-4 ± 3.0 × 10-4. Stability assessment revealed a thermal tolerance up to 60 ˚C, wide range pH stability (3-10) and the ability to withstand shearing stress up to 250 rpm. HK6 shows no cytotoxicity against oral epithelial cells up to 1012 PFU/ml. Genomic analysis revealed a Strabovirus with total size of 177,845 bp that is free from known resistance and virulence genes. Finally, HK6 pretreatment of raw chicken, chicken nuggets and ready-made cheese salad shows a reduced bacterial count up to 4.6, 2.96 and 2.81 log-units, respectively. Moreover, combing HK6 with sodium nitrite further improved the antibacterial activity in both raw chicken and chicken nuggets without significant enhancement in case of cheese salad. CONCLUSION: Enterobacter bacteriophage vB_EclM_HK6 presents a safe and effective approach for controlling E. cloacae contaminating stored chicken food samples. Moreover, they could be combined with a reduced concentrations of sodium nitrite to improve the killing capacity.

Nitric oxide donors rescue metabolic and mitochondrial dysfunction in obese Alzheimer's model.

Reduced nitric oxide (NO) bioavailability is a pathological link between obesity and Alzheimer's disease (AD). Obesity-associated metabolic and mitochondrial bioenergetic dysfunction are key drivers of AD pathology. The hypothalamus is a critical brain region during the development of obesity and dysfunction is an area implicated in the development of AD. NO is an essential mediator of blood flow and mitochondrial bioenergetic function, but the role of NO in obesity-AD is not entirely clear. We investigated diet-induced obesity in female APPswe/PS1dE9 (APP) mouse model of AD, which we treated with two different NO donors (sodium nitrite or L-citrulline). After 26 weeks of a high-fat diet, female APP mice had higher adiposity, insulin resistance, and mitochondrial dysfunction (hypothalamus) than non-transgenic littermate (wild type) controls. Treatment with either sodium nitrite or L-citrulline did not reduce adiposity but improved whole-body energy expenditure, substrate oxidation, and insulin sensitivity. Notably, both NO donors restored hypothalamic mitochondrial respiration in APP mice. Our findings suggest that NO is an essential mediator of whole-body metabolism and hypothalamic mitochondrial function, which are severely impacted by the dual insults of obesity and AD pathology.

Impaired Endothelium-Dependent Vasodilation and Increased Levels of Soluble Fms-like Tyrosine Kinase-1 Induced by Reduced Uterine Perfusion Pressure in Pregnant Rats: Evidence of Protective Effects with Sodium Nitrite Treatment in Preeclampsia.

Preeclampsia (PE) is a hypertensive disorder of pregnancy and is associated with increases in soluble fms-like tyrosine kinase-1 (sFlt-1) and reductions in nitric oxide (NO) levels. Placental ischemia and hypoxia are hypothesized as initial pathophysiological events of PE. Nitrite (NO metabolite) may be recycled back to NO in ischemic and hypoxic tissues. Therefore, this study examined the sodium nitrite effects in an experimental model of PE. Pregnant rats received saline (Preg group) or sodium nitrite (Preg + Na-Nitrite group). Pregnant rats submitted to the placental ischemia received saline (RUPP group) or sodium nitrite (RUPP + Na-Nitrite group). Blood pressure, placental and fetal weights, and the number of pups were recorded. Plasma levels of NO metabolites and sFlt-1 were also determined. Vascular and endothelial functions were also measured. Blood pressure, placental and fetal weights, the number of pups, NO metabolites, sFlt-1 levels, vascular contraction, and endothelium-dependent vasodilation in the RUPP + Na-Nitrite rats were brought to levels comparable to those in Preg rats. In conclusion, sodium nitrite may counteract the reductions in NO and increases in sFlt-1 levels induced by the placental ischemia model of PE, thus suggesting that increased blood pressure and vascular and endothelial dysfunctions may be attenuated by sodium nitrite-derived NO.

The hemodynamic response to nitrite is acute and dependent upon tissue perfusion.

In the vasculature, nitric oxide (NO) is produced in the endothelium by endothelial nitric oxide synthase (eNOS) and is critical for the regulation of blood flow and blood pressure. Blood flow may also be regulated by the formation of nitrite-derived NO catalyzed by hemoproteins under hypoxic conditions. We sought to investigate whether nitrite administration may affect tissue perfusion and systemic hemodynamics in WT and eNOS knockout mice. We found that global eNOS KO mice show decreased tissue perfusion compared to WT mice by using laser speckle contrast imaging. To study both the acute and long-term effects of sodium nitrite (0, 0.1, 1, and 10 mg/kg) on peripheral blood flow and systemic blood pressure, a bolus of nitrite was delivered intraperitoneally every 24 h over 4 consecutive days. We found that nitrite administration resulted in a dose-dependent and acute increase in peripheral blood flow in eNOS KO mice but had no effects in WT mice. The nitrite induced changes in tissue perfusion were transient, as determined by intraindividual comparisons of tissue perfusion 24-h after injection. Accordingly, 10 mg/kg sodium nitrite acutely decreased blood pressure in eNOS KO mice but not in WT mice as determined by invasive Millar catheterization. Interestingly, we found the vasodilatory effects of nitrite to be inversely correlated to baseline tissue perfusion. These results demonstrate the nitrite acutely recovers hypoperfusion and hypertension in global eNOS KO mice and suggest the vasodilatory actions of nitrite are dependent upon tissue hypoperfusion.

Redox-Active Compounds in the Therapy of Drug-Resistant Murine Leukemia P388 Strains.

The efficiency of combinations of cytostatics cisplatin and adriamycin with antioxidant sodium 3-(3'-tert-butyl-4-hydroxyphenyl)propyl thiosulfate (TS-13), and nitric oxide (NO) donor NaNO2 was evaluated on two drug-resistant strains of leukemia P388 with changed redox-status of cells. Simultaneous use of both NO donor and TS-13 in combinations with the cytostatics did not increase the efficiency of therapy. In addition, antioxidant activity of TS-13, NaNO2, and their combinations was studied by the method of luminol-dependent chemiluminescence on the model systems with the use of the homogenized cells of sensitive strain and two drug-resistant strains of leukemia P388. It was shown that TS-13 and NO donor produced opposite effects: TS-13 decreased, while NO donor increased the content of free radicals in the model system. Combinations of antioxidant TS-13 and NO donor should be used with consideration for the redox-status of tumor treated.

Impact of chlorogenic acid on submandibular salivary gland and liver of albino rats exposed to sodium nitrite.

AIM: The aim of the present study is to show how sodium nitrite alters the histology of submandibular salivary glands and livers of Albino rats, as well as how chlorogenic acid may have therapeutic benefits. METHODS: A sample size of thirty male Sprague Dawley Albino rats weighing between 100 and 150 g (5-6 weeks old) was randomly allocated into 3 equal groups. Group I: rats were used as controls and were given phosphate buffer solution, whereas Group II: rats were given an 80 mg/kg sodium nitrites (SN) daily dissolved in distilled water. The rats in Group III were given a daily dose of 80 mg/kg SN dissolved in distilled water and after 6 hours each rat received 50 mg/mL freshly prepared chlorogenic acid (CGA) every other day. For 12 weeks, all treatment modalities will be administered orally, every day. After the experiment, all rats were euthanized. Samples from salivary glands and livers were processed and stained with H&E and interleukin 6 (IL 6). Malondialdehyde (MDA) and superoxide dismutase (SOD) enzymes were detected using an ELISA assay. RESULTS: Groups III had nearly comparable findings to Group I regarding histological pattern with normal submandibular glands and livers features. Group III salivary gland treated with CGA exhibited higher SOD levels (20.60±4.81 U/g) in comparison to the SN group, and lower MDA levels (111.58±28.28 nmol/mg) in comparison to the SN treated samples. In comparison to the SN group, CGA treatment significantly reduced MDA levels in liver samples (167.56±21.17 nmol/mg) and raised SOD (30.85±6.77 U/g). CONCLUSIONS: Chlorogenic acid has a protective effect against salivary gland and liver toxicity induced by SN in rats. This was mediated via the anti-inflammatory and antioxidative properties of CGA and the restoration of oxidant/antioxidant balance in rat salivary gland and liver.

Growth and no-growth boundary of Clostridium perfringens in cooked cured meats - A logistic analysis and development of critical control surfaces using a solid growth medium.

This study was conducted to evaluate the effect of sodium nitrite (NaNO2, 100-200 ppm), sodium erythorbate (SE, 0-547 ppm), sodium tripolyphosphate (STPP, 0-0.5 %), and sodium chloride (NaCl, 2-3 %) on growth of C. perfringens using a solid growth medium and to develop a growth/no-growth boundary (critical control surface, or CCS) to prevent its growth in cooked cured meat under the optimal temperature condition. Melted Shahidi Ferguson Perfringens (SFP) agar, inoculated with a 3-strain spore cocktail and mixed with NaNO2, SE, STPP, and NaCl according to a Box-Behnken response surface experimental design, was dispersed in 96-well microplates and incubated anaerobically in an incubator programmed to remain at 4 °C for 24 h, heat to 80 °C in 1.75 h, quickly (0.5 h) cool to 46 °C (optimum temperature), and then maintain at 46 °C overnight. The plates were examined optically and visually for colony formation. Any well free of growth was designated as no-growth. Logistic regression was used to analyze the growth probability (P) as affected by NaNO2, SE, STPP, and NaCl and define a CSS as meeting the criterion of P < 1/96. The results showed that STPP and the interactions of SE with NaNO2 and NaCl could reduce the growth probability of C. perfringens in SFP agar. The validation of CCS with ground beef showed an accuracy of 96.3 % for no growth of C. perfringens in the inoculated samples. The results of this study proved that cured meat can be formulated with proper combinations of NaNO2, SE, STPP, and NaCl to prevent the growth of C. perfringens even under the optimum temperature condition, thus preventing food poisoning caused by the growth of this microorganism.

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.

The bacillithiol pathway is required for biofilm formation in Staphylococcus aureus.

Staphylococcus aureus is a major human pathogen that can cause infections that range from superficial skin and mucosal infections to life threatening disseminated infections. S. aureus can attach to medical devices and host tissues and form biofilms that allow the bacteria to evade the host immune system and provide protection from antimicrobial agents. To counter host-generated oxidative and nitrosative stress mechanisms that are part of the normal host responses to invading pathogens, S. aureus utilizes low molecular weight (LMW) thiols, such as bacillithiol (BSH). Additionally, S. aureus synthesizes its own nitric oxide (NO), which combined with its downstream metabolites may also protect the bacteria against specific host responses. We have previously shown that LMW thiols are required for biofilm formation in Mycobacterium smegmatis and Pseudomonas aeruginosa. Here, we show that the S. aureus bshC mutant strain, which is defective in the last step of the BSH pathway and lacks BSH, is impaired in biofilm formation. We also identify a possible S-nitrosobacillithiol reductase (BSNOR), similar in sequence to an S-nitrosomycothiol reductase found in M. smegmatis and show that the putative S. aureus bsnoR mutant strain has reduced levels of BSH and decreased biofilm formation. Our studies also show that NO plays an important role in biofilm formation and that acidified sodium nitrite severely reduces biofilm thickness. These studies provide insight into the roles of oxidative and nitrosative stress mechanisms on biofilm formation and indicate that BSH and NO are key players in normal biofilm formation in S. aureus.

Emulsified and Nanoemulsified Essential Oils in the Control of Clostridium botulinum and Clostridium sporogenes in Mortadella.

Clostridium botulinum is a foodborne pathogen responsible for severe neuroparalytic disease associated with the ingestion of pre-formed toxin in food, with processed meats and canned foods being the most affected. Control of this pathogen in meat products is carried out using the preservative sodium nitrite (NaNO2), which in food, under certain conditions, such as thermal processing and storage, can form carcinogenic compounds. Therefore, the objective was to use nanoemulsified essential oils (EOs) as natural antimicrobial agents, with the aim of reducing the dose of NaNO2 applied in mortadella. The antimicrobial activity of nanoemulsions prepared with mixtures of EOs of garlic, clove, pink pepper, and black pepper was evaluated on endospores and vegetative cells of C. botulinum and Clostridium sporogenes (surrogate model) inoculated in mortadella prepared with 50 parts per million NaNO2. The effects on the technological (pH, water activity, and color) and sensory characteristics of the product were also evaluated. The combinations of EOs and their nanoemulsions showed sporicidal effects on the endospores of both tested microorganisms, with no counts observed from the 10th day of analysis. Furthermore, bacteriostatic effects on the studied microorganisms were observed. Regarding the technological and sensorial characteristics of the product, the addition of the combined EOs had a negative impact on the color of the mortadella and on the flavor/aroma. Despite the strong commercial appeal of adding natural preservatives to foods, the effects on flavor and color must be considered. Given the importance of controlling C. botulinum in this type of product, as well as the reduction in the amount of NaNO2 used, this combination of EOs represents a promising antimicrobial alternative to this preservative, encouraging further research in this direction.

A simple and efficient alginate hydrogel combined with surface-enhanced Raman spectroscopy for quantitative analysis of sodium nitrite in meat products.

Sodium nitrite is a commonly used preservative and color protectant in the food industry. Conventional analytical methods are highly susceptible to food matrix interference, time-consuming and costly. In this study, the ion cross-linking method was employed to prepare alginate hydrogel substrates, and phenosafranin was chosen as a single-molecule probe to analyze sodium nitrite. Our investigation centered on elucidating the effects of alginate and cross-linking ion concentrations on Raman signal characteristics. The optimal Raman response was observed in the precursor solution with 1% sodium alginate and 0.1 mol L-1 cross-linking ions. The relative standard deviations (RSDs) of the feature peaks from the three substrate batches ranged from 1.22% to 16.30%, attesting the robustness and consistency of the substrates. The signal reduction of the substrates after a four-week storage period remained below 10%, indicating that the substrates had good reproducibility and stability. The limits of detection (LODs) for sodium nitrite in extracts from cured meat, luncheon meat, and sliced ham were determined to range from 3.75 mg kg-1 to 8.11 mg kg-1, with low interference from the food matrix. The support vector machine algorithm was utilized to train and predict the data, which proved to be more accurate (98.6%-99.8% recovery) than the traditional linear regression model (81.9%-112.7% recovery) in predicting the spiked samples. The application of hydrogel-based surface-enhanced Raman spectroscopy (SERS) substrates for nitrite detection in food, combined with machine learning for regression prediction in data processing, collectively augmented the potential of SERS technology in the field of food analysis.

Sodium Nitrite Attenuates Reduced Activity of Vascular Matrix Metalloproteinase-2 and Vascular Hyper-Reactivity and Increased Systolic Blood Pressure Induced by the Placental Ischemia Model of Preeclampsia in Anesthetized Rats.

Preeclampsia is a maternal hypertension disorder associated with vascular dysfunction and fetal and placental growth restrictions. Placental ischemia is suggested as the primary trigger of preeclampsia-associated impairments of both endothelium-derived nitric oxide (NO) and the vascular activity of extracellular matrix metalloproteinase-2 (MMP-2). Reduced uteroplacental perfusion pressure (RUPP) is a placental ischemia model of preeclampsia. Reduction of sodium nitrite to NO may occur during ischemic conditions. However, sodium nitrite effects in the RUPP model of preeclampsia have not yet been investigated. Pregnant rats were divided into four groups: normotensive pregnant rats (Norm-Preg), pregnant rats treated with sodium nitrite (Preg + Nitrite), preeclamptic rats (RUPP), and preeclamptic rats treated with sodium nitrite (RUPP + Nitrite). Maternal blood pressure and fetal and placental parameters were recorded. Vascular function, circulating NO metabolites, and the gelatinolytic activity of vascular MMP-2 were also examined. Sodium nitrite attenuates increased blood pressure, prevents fetal and placental weight loss, counteracts vascular hyper-reactivity, and partially restores NO metabolites and MMP-2 activity. In conclusion, sodium nitrite reduction to NO may occur during RUPP-induced placental ischemia, thereby attenuating increased blood pressure, fetal and placental growth restriction, and vascular hyper-reactivity associated with preeclampsia and possibly restoring NO and MMP-2 activity, which underlie the blood pressure-lowering effects.

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.

Nitrite lowers the oxygen cost of ATP supply in cultured skeletal muscle cells by stimulating the rate of glycolytic ATP synthesis.

Dietary nitrate lowers the oxygen cost of human exercise. This effect has been suggested to result from stimulation of coupling efficiency of skeletal muscle oxidative phosphorylation by reduced nitrate derivatives. In this paper, we report the acute effects of sodium nitrite on the bioenergetic behaviour of cultured rat (L6) myocytes. At odds with improved efficiency of mitochondrial ATP synthesis, extracellular flux analysis reveals that a ½-hour exposure to NaNO2 (0.1-5 µM) does not affect mitochondrial coupling efficiency in static myoblasts or in spontaneously contracting myotubes. Unexpectedly, NaNO2 stimulates the rate of glycolytic ATP production in both myoblasts and myotubes. Increased ATP supply through glycolysis does not emerge at the expense of oxidative phosphorylation, which means that NaNO2 acutely increases the rate of overall myocellular ATP synthesis, significantly so in myoblasts and tending towards significance in contractile myotubes. Notably, NaNO2 exposure shifts myocytes to a more glycolytic bioenergetic phenotype. Mitochondrial oxygen consumption does not decrease after NaNO2 exposure, and non-mitochondrial respiration tends to drop. When total ATP synthesis rates are expressed in relation to total cellular oxygen consumption rates, it thus transpires that NaNO2 lowers the oxygen cost of ATP supply in cultured L6 myocytes.

Effect of Biofunctional Green Synthesized MgO-Nanoparticles on Oxidative-Stress-Induced Tissue Damage and Thrombosis.

The present study describes the green biofunctional synthesis of magnesium oxide (MgO) nanoparticles using the aqueous Tarenna asiatica fruit extract. The characterization of Tarenna asiatica fruit extract MgO nanoparticles (TAFEMgO NPs) was achieved by X-ray powder diffraction, UV-Vis spectroscopy, FTIR, TEM, SEM, and energy-dispersive X-ray diffraction. TAFEMgO NPs scavenged the DPPH free radicals with an IC50 value of 55.95 µg/µL, and it was highly significant compared to the standard. To authenticate the observed antioxidant potential of TAFEMgO NPs, oxidative stress was induced in red blood cells (RBC) using sodium nitrite (NaNO2). Interestingly, TAFEMgO NPs ameliorated the RBC damage from oxidative stress by significantly restoring the stress parameters, such as the protein carbonyl content (PCC), lipid peroxidation (LPO), total thiol (TT), super-oxide dismutase (SOD), and catalase (CAT). Furthermore, oxidative stress was induced in-vivo in Sprague Dawley female rats using diclofenac (DFC). TAFEMgO NPs normalized the stress parameters in-vivo and minimized the oxidative damage in tissues. Most importantly, TAFEMgO NPs restored the function and architecture of the damaged livers, kidneys, and small intestines by regulating biochemical parameters. TAFEMgO NPs exhibited an anticoagulant effect by increasing the clotting time from 193 s in the control to 885 s in the platelet rich plasma. TAFEMgO NPs prolonged the formation of the clot process in the activated partial thromboplastin time and the prothrombin time, suggest the effective involvement in both intrinsic and extrinsic clotting pathways of the blood coagulation cascade. TAFEMgO NPs inhibited adenosine di-phosphate (ADP)-induced platelet aggregation. TAFEMgO NPs did not show hemolytic, hemorrhagic, and edema-inducing properties at the tested concentration of 100 mg/kgbody weight, suggesting its non-toxic property. In conclusion, TAFEMgO NPs mitigates the sodium nitrite (NaNO2)- and diclofenac (DFC)-induced stress due to oxidative damage in both in vitro and in vivo experimental models.

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.

Targeted Anti-Cancer Provascular Therapy Using Ultrasound, Microbubbles, and Nitrite to Increase Radiotherapy Efficacy.

Hypoxia is an important mechanism of resistance to radiation therapy in many human malignancies including prostate cancer. It has been recently shown that ultrasound targeted microbubble cavitation (UTMC) can increase blood perfusion in skeletal muscle by triggering nitric oxide signaling. Interestingly, this effect was amplified with a sodium nitrite coinjection. Since sodium nitrite has been shown to synergize with radiotherapy (RT), we hypothesized that UTMC with a sodium nitrite coinjection could further radiosensitize solid tumors by increasing blood perfusion and thus reduce tumor hypoxia. We evaluated (1) the ability of UTMC with and without nitrite to increase perfusion in muscle (mouse hindlimbs) and human prostate tumors using different pulse lengths and pressure; (2) the efficacy of this approach as a provascular therapy given directly before RT in the human prostate subcutaneous xenografts PC3 tumor model. Using long pulses with various pressures, in muscle, the provascular response following UTMC was strong (6.61 ± 4.41-fold increase in perfusion post-treatment). In tumors, long pulses caused an increase in perfusion (2.42 ± 1.38-fold) at lower mechanical index (MI = 0.25) but not at higher MI (0.375, 0.5, and 0.750) when compared to control (no UTMC). However, when combined with RT, UTMC with long pulses (MI = 0.25) did not improve tumor growth inhibition. With short pulses, in muscle, the provascular response following UTMC (SONOS) + nitrite was strong (13.74 ± 8.60-fold increase in perfusion post-treatment). In tumors, UTMC (SONOS) + nitrite also caused a provascular response (1.94 ± 1.20-fold increase in perfusion post-treatment) that lasted for at least 10 min, but not with nitrite alone. Interestingly, the blunted provascular response observed for long pulses at higher MI without nitrite was reversed with the addition of nitrite. UTMC (SONOS) with and without nitrite caused an increase in perfusion in tumors. The provascular response observed for UTMC (SONOS) + nitrite was confirmed by histology. Finally, there was an improved growth inhibition for the 8 Gy RT dose + nitrite + UTMC group vs 8 Gy RT + nitrite alone. This effect was not significant with mice treated by UTMC + nitrite and receiving doses of 0 or 2 Gy RT. In conclusion, UTMC + nitrite increased blood flow leading to an increased efficacy of higher doses of RT in our tumor model, warranting further study of this strategy.

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.

Sodium hydrosulfide has no additive effects on nitrite-inhibited renal gluconeogenesis in type 2 diabetic rats.

OBJECTIVE: Increased renal and hepatic gluconeogenesis are important sources of fasting hyperglycemia in type 2 diabetes (T2D). The inhibitory effect of co-administration of sodium nitrite and sodium hydrosulfide (NaSH) on hepatic but not renal gluconeogenesis has been reported in rats with T2D. The present study aimed to determine the effects of co-administration of sodium nitrite and NaSH on the expression of genes involved in renal gluconeogenesis in rats with T2D. METHODS: T2D was induced by a combination of a high-fat diet and low-dose streptozotocin (30 mg/kg). Male Wistar rats were divided into 5 groups (n = 6/group): Control, T2D, T2D + nitrite, T2D + NaSH, and T2D + nitrite+NaSH. Nitrite and NaSH were administered for nine weeks at a dose of 50 mg/L (in drinking water) and 0.28 mg/kg (daily intraperitoneal injection), respectively. Serum levels of urea and creatinine, and mRNA expressions of PEPCK, G6Pase, FBPase, PC, PI3K, AKT, PGC-1α, and FoxO1 in the renal tissue, were measured at the end of the study. RESULTS: Nitrite decreased mRNA expression of PEPCK by 39%, G6Pase by 43%, FBPase by 41%, PC by 63%, PGC-1α by 45%, and FoxO1 by 27% in the renal tissue of rats with T2D; co-administration of nitrite and NaSH further decreases FoxO1, while had no additive effects on the tissue expression of the other genes. In addition, nitrite+NaSH decreased elevated serum urea levels by 58% and creatinine by 37% in rats with T2D. CONCLUSION: The inhibitory effect of nitrite on gluconeogenesis in T2D rats is at least in part due to decreased mRNA expressions of renal gluconeogenic genes. Unlike effects on hepatic gluconeogenesis, co-administration of nitrite and NaSH has no additive effects on genes involved in renal gluconeogenesis in rats with T2D.