High Levels Of Nitrite Research Articles

Effect of carbon to nitrogen (C:N) ratio on nitrogen removal from shrimp production waste water using sequencing batch reactor

The United States Marine Shrimp Farming Program (USMSFP) introduced a new technology for shrimp farming called recirculating raceway system. This is a zero-water exchange system capable of producing high-density shrimp yields. However, this system produces wastewater characterized by high levels of ammonia, nitrite, and nitrate due to 40% protein diet for the shrimp at a high density of 1,000 shrimp per square meter. The high concentrations of nitrate and nitrite (greater than 25ppm) are toxic to shrimp and cause high mortality. So treatment of this wastewater is imperative in order to make shrimp farming viable. One simple method of treating high-nitrogen wastewater is the use of a sequencing batch reactor (SBR). An SBR is a variation of the activated sludge process, which accomplishes many treatment events in a single reactor. Removal of ammonia and nitrate involved nitrification and denitrification reactions by operating the SBR aerobically and anaerobically in sequence. Initial SBR operation successfully removed ammonia, but nitrate concentrations were too high because of carbon limitation in the shrimp production wastewater. An optimization study revealed the optimum carbon to nitrogen (C:N) ratio of 10:1 for successful removal of all nitrogen species from the wastewater. The SBR operated with a C:N ratio of 10:1 with the addition of molasses as carbon source successfully removed 99% of ammonia, nitrate, and nitrite from the shrimp aquaculture wastewater within 9days of operation.

Effect of nitrite on growth and microcystins production of Microcystis aeruginosa PCC7806

As a common pollutant, nitrite concentrations can approach 15 mg NO2−-N L−1 in some aquatic systems. Microcystis aeruginosa blooms are common and widespread in eutrophic freshwater bodies. In this study, M. aeruginosa was exposed to nitrite concentrations ranging from 0 to 15 mg NO2−-N L−1, and the responses of M. aeruginosa were investigated. The specific growth rates, maximum cell densities, light-saturated photosynthetic rates (Pmchla), dark respiration rates (Rdchla), and apparent photosynthetic efficiencies (αchla) showed a significant decline with nitrite concentrations increasing. Electrical conductivity and malondialdehyde contents investigation revealed cell membrane damage and apparent leakage of intracellular contents under high nitrite level conditions due to oxidative stress enhancement. Intracellular microcystin (MC)-LR content reached the highest value at 10 mg NO2−-N L−1; however, extracellular MC-LR contents showed a continuous increase until 15 mg NO2−-N L−1 owing to the increasing leakage of intracellular contents. These results elucidated that the high-level nitrite inhibited M. aeruginosa growth by rising oxidative stress, damaging cell membrane, and reducing photosynthesis. However, the moderate increase in nitrite concentrations promoted toxin production and release of toxin.

Evaluation of nitrifying bacteria product to improve nitrification efficacy in recirculating aquaculture systems

Recirculating aquaculture systems (RASs) rely on nitrification to convert toxic ammonia and nitrite to less toxic nitrate. Nitrification is accomplished using biofilters with nitrifying bacteria and can be inefficient in biofilters that are new or that have been compromised due to stressors. Failure in a biofilter can result in very high levels of ammonia or nitrite, both of which are toxic to aquatic animals and can result in health issues, suppressed growth, and mortalities. For these reasons, a commercially available nitrifying bacterial product (Pond Protect-L®, Novozymes Biologicals Inc., Salem, VA, US) was tested in both a controlled pilot-scale experiment and within production-scale RAS that were experiencing elevated levels of nitrite. Juvenile Pacific white shrimp (Litopenaeus vannamei) were used as the aquatic species in both the pilot- and production-scale systems. In the pilot-scale experiment, three aquaria received no product (control) and three systems received bacterial product using dosing specification as recommended by the manufacturer. Control systems experienced significantly (P<0.05) higher levels of ammonia and nitrite compared to the tanks that received bacterial product. Quantitative PCR was used as tool to verify the presence of nitrifying bacteria (Nitrobacter winogradskyi strain in the bacterial product) in the systems that received bacterial product. Meanwhile, none of these nitrifiers were detected in the control systems. Similar nitrification benefits were noted in the production systems that received bacterial product. More specifically, improved nitrite-oxidation was observed. Within 2 days of the addition of bacterial product, nitrite levels began to decrease in the production systems and after a steady decrease remained within safe limits for the entire shrimp culture period.

Effects of Ammonium and Nitrite on Growth and Competitive Fitness of Cultivated Methanotrophic Bacteria

The effects of nitrite and ammonium on cultivated methanotrophic bacteria were investigated. Methylomicrobium album ATCC 33003 outcompeted Methylocystis sp. strain ATCC 49242 in cultures with high nitrite levels, whereas cultures with high ammonium levels allowed Methylocystis sp. to compete more easily. M. album pure cultures and cocultures consumed nitrite and produced nitrous oxide, suggesting a connection between denitrification and nitrite tolerance.

Effects of eNOS polymorphisms on nitric oxide formation in healthy pregnancy and in pre-eclampsia

Pre-eclampsia (PE) is associated with decreased nitric oxide (NO) formation. However, no previous study has examined whether genetic variations in the endothelial NO synthase (eNOS) affect this alteration. We hypothesized that PE decreases NO formation depending on eNOS polymorphisms. We examined how three eNOS polymorphisms [T-786C, rs2070744; Glu298Asp, rs1799983; 27 bp variable number of tandem repeats (VNTR) in intron 4] affect plasma nitrite concentrations in 205 pregnant women [107 healthy pregnant (HP) and 98 PE]. Genotypes were determined and eNOS haplotypes were inferred using the PHASE 2.1 program. The plasma nitrite concentrations were determined using an ozone-based chemiluminescence assay. The Glu298Asp polymorphism had no effects on the plasma nitrite concentrations. Higher nitrite levels were found in HP women with the CC versus TT genotype for the T-786C polymorphism (277.9 +/- 19.5 versus 140.6 +/- 8.2 nM; P < 0.05). Lower nitrite levels were found in healthy women with the 4a4a versus 4b4b genotype for the VNTR polymorphism (95.1 +/- 3.3 versus 216.1 +/- 16.8 nM; P < 0.05). No effects of genotypes were found in PE women (all P > 0.05). The 'C Glu b' haplotype was more frequent in the HP group than in the PE group (20 versus 5; P = 0.0044). This haplotype was associated with higher nitrite concentrations than the other haplotypes in healthy pregnancies (P < 0.05). No differences in nitrite concentrations were found among PE women with different eNOS haplotypes (P > 0.05). These findings indicate that eNOS polymorphisms affect endogenous NO formation in normal pregnancy, but not in PE, and that the 'C Glu b' haplotype may protect against the development of PE by increasing endogenous NO formation.

Growth parameters of microalgae tolerant to high levels of carbon dioxide in batch and continuous‐flow photobioreactors

Microalgae can be cultured in photobioreactors to sequester carbon dioxide and produce potentially valuable biomaterials. The goal of the present study was to identify and utilize microalgal strains that are capable of tolerating up to 20% CO2 (gas phase) concentrations under variable light or flue‐gas blend conditions and reactor configurations to produce biomass. Scenedesmus sp. and Chlorella sp., both cultured from a Sonoran desert mineral spring, grew well and tolerated exposure to a gas mixture containing up to 20% CO2 applied continuously in batch reactors to the culture. Experiments were conducted with simulated coal‐powered acidic flue gases containing SOx/NOx at concentrations of 200 to 350 ppmV. Microalgae did not grow well without pH control, and high levels (>250 mM) of nitrite or sulphite in the liquid media inhibited algal growth. Pseudo steady‐state experiments were also conducted using helical tubular and flat‐plate photobioreactors with continuous flow (water and gas) and with artificial or natural sunlight. With a 2 d hydraulic residence time (HRT), the helical tubular photobioreactor produced 0.50 ± 0.11 g C d−1 (0.056 ± 0.012 g C L−1 d−1) dry‐weight cell mass during continuous fluorescent‐lamp irradiance and 0.048 ± 0.018 g C L−1 d−1 during 12 h light/darkness cycling. The flat‐plate photobioreactor (2 d HRT) produced 0.42 ± 0.28 g C L−1 d−1 with artificial lighting and with natural sunlight; a 4 d HRT produced 0.14 ± 0.02 g C L−1 d−1. Reactor modelling indicated that a threshold of reactor size (i.e. HRT) and reactor depth (path‐length of light) exists based upon the optical density of the cells in the water column and their growth rates.

Induced sputum nitrites correlate with FEV1in children with cystic fibrosis

To determine the difference in the levels of nitrites in induced sputum of children with cystic fibrosis (CF) and controls. Furthermore, to evaluate the association between induced sputum nitrites and lung function in children with CF. Nitrites, cell differentials, white blood cell count, were estimated in induced sputum of 20 children with CF and 10 age-matched healthy controls. Nitrites in induced sputum samples were measured using the Greiss assay. Lung function was ascertained by spirometry. We observed high levels of nitrites in CF (184.8 +/- 11.07 microM/L) versus controls (56.4 +/- 5.7 microM/L) (p < 0.01). A positive correlation between neturophil percent and nitrites, white blood cell count and nitrites (p < 0.05) in children with CF was observed. Sputum nitrites correlated negatively with FEV(1) (p < 0.05) in children with CF. Induced sputum nitrite could serve as a useful non invasive marker for assessing the degree of inflammation in the airways of children with CF.

Production of hydrogen peroxide and nitric oxide following introduction of nitrate and nitrite into wheat leaf apoplast

Infiltration of wheat (Triticum aestivum L.) seedling leaves with excess of nitrate, nitrite, or the NO donor sodium nitroprusside leads to increase both in content of hydroperoxide and activity of peroxidase and decrease in superoxide dismutase (SOD) activity in the leaf apoplast. Polymorphism of extracellular peroxidases and the presence of Cu/Zn-SOD have been shown in apoplast. Using an ESR assay, a considerable increase in the level of NO following infiltration of leaf tissues with nitrite has been demonstrated. These data suggest development of both oxidative and nitrosative stresses in leaves exposed to high levels of nitrate or nitrite. A possible interplay of NO and reactive oxygen species in plant cells is discussed.

Differential Reactivity of Purified Bioactive Coffee Furans, Cafestol and Kahweol, with Acidic Nitrite: Product Characterization and Factors Controlling Nitrosation Versus Ring-Opening Pathways

Cafestol and kahweol, coffee-specific furan diterpenes, are believed to cause various physiological effects in human subjects, including an increase in cholesterol and plasma triacylglycerol levels as well as cancer chemopreventive effects. Despite the increasing interest in these compounds raised by the diverse range of biological activities, their reaction behavior and degradation pathways under physiologically relevant conditions remain uncharted. Herein, we report a detailed investigation of the structural modifications suffered by cafestol and kahweol in the presence of acidic nitrite under conditions mimicking those occurring in the stomach during digestion as well as by action of other oxidants. Prior to the chemical study, an isolation procedure for kahweol from green coffee beans was developed based on Soxhlet extraction followed by preparative HPLC. Preliminary experiments showed that kahweol is much more reactive than cafestol toward nitrite at pH 3, as evidenced by inhibition experiments with the 2,3-diaminonaphthalene assay as well as by product analysis in coffee extracts. When exposed to equimolar nitrite in phosphate buffer, pH 3, kahweol gave as a main product the ring-opened dicarbonyl derivative 1. Under more forcing conditions, cafestol reacted as well to give a main nitrogenous product identified as the 1-hydroxy-2-pyrrolinone 2. It is concluded that the conjugated double bond in kahweol is a critical structural element, increasing the susceptibility of the furan ring to protonation rather than nitrosation and favoring ring-opening routes driven by the irreversible oxidation steps. These results offer a useful background to assess the effects of coffee-specific lipids in association with abnormally high nitrite levels from the diet.

Cerebrospinal Fluid Nitric Oxide Levels in Subacute Sclerosing Panencephalitis

Oxidative damage plays a role in neurodegenerative diseases. Levels of cerebrospinal fluid nitrite and nitrate levels (oxidation products that provide an indirect estimation of nitric oxide) were investigated in relation to clinical and laboratory features in subacute sclerosing panencephalitis (n = 47) and age-matched control (n = 43) groups. Significantly decreased levels of nitrite (median, 4.91 micromol/L) and nitrate (median, 6.14 micromol/L) were found in the patients. Nitrite and nitrate levels did not correlate with clinical or laboratory findings, except for presence of myoclonus. Cerebrospinal fluid nitrite levels of subacute sclerosing panencephalitis patients without myoclonic jerks were significantly higher than in those with myoclonus (median, 15.63 vs 4.34 micromol/L, respectively). The higher levels of nitrite in these patients can be explained by short disease duration and early stages of disease. Nitrate levels in subacute sclerosing panencephalitis patients with myoclonus (median, 9.26 micromol/L) were higher than in those without myoclonus (median, 4.25 micromol/L). Microbleeding resulting in conversion of nitrite to nitrate and increased production of superoxide can be suggested as possible mechanisms underlying these findings.

Occurrence of High In-Stream Nitrite Levels in a Temperate Region Agricultural Watershed

This study is the first to report nitrite occurrence and variability in surface water across an agricultural watershed in the province of Quebec, Canada. Nitrite (NO2 −) concentrations were monitored across a 2.4 km2 watershed during 2 years. Water samples were collected at the outlet from April to November and in three contrasted stream branches (six stations) during three hydrological regimes (summer low water levels, fall recharge, and snowmelt). Our study clearly demonstrates that NO2 − levels observed at the outlet are not representative of NO2 − variability across the micro-watershed. Surface water collected in cropped areas presents high NO2 − concentrations during summer low water levels, often exceeding guidelines for aquatic life, with NO2 − means ranging from 0.022 to 0.107 mg N L−1 and maximum value reaching 0.156 mg N L−1. Furthermore, the two stream branches in cropped area have demonstrated significant differences in NO2 − concentrations and other water quality parameters. The importance of groundwater discharge to streams in the micro-watershed Bras d’Henri may potentially generate different in-stream sources of NO2 − and water quality parameters. However, further studies are essential to determine sources and processes related to in-stream NO2 − peaks.

Biological treatment of shrimp production wastewater

Over the last few decades, there has been an increase in consumer demand for shrimp, which has resulted in its worldwide aquaculture production. In the United States, the stringent enforcement of environmental regulations encourages shrimp farmers to develop new technologies, such as recirculating raceway systems. This is a zero-water exchange system capable of producing high-density shrimp yields. The system also produces wastewater characterized by high levels of ammonia, nitrate, nitrite, and organic carbon, which make waste management costs prohibitive. Shrimp farmers have a great need for a waste management method that is effective and economical. One such method is the sequencing batch reactor (SBR). A SBR is a variation of the activated sludge biological treatment process. This process uses multiple steps in the same reactor to take the place of multiple reactors in a conventional treatment system. The SBR accomplishes equalization, aeration, and clarification in a timed sequence in a single reactor system. This is achieved through reactor operation in sequences, which includes fill, react, settle, decant, and idle. A laboratory scale SBR was successfully operated using shrimp aquaculture wastewater. The wastewater contained high concentrations of carbon and nitrogen. By operating the reactors sequentially, namely, aerobic and anoxic modes, nitrification and denitrification were achieved as well as removal of carbon. Ammonia in the waste was nitrified within 4 days. The denitrification of nitrate was achieved by the anoxic process, and 100% removal of nitrate was observed within 15 days of reactor operation.

Alkyl nitrate photochemical production rates in North Pacific seawater

Low molecular weight alkyl nitrates are produced in surface seawater from the photochemically initiated reaction of NO with organic peroxy radicals. In this study, methyl, ethyl, isopropyl, and n-propyl nitrate photochemical production rates were determined from shipboard incubation experiments. Surface water samples from a variety of different North Pacific water masses were irradiated in sunlight, with and without added nitrite. Production was observed in some, but not all unmodified waters. Production rates increased with increasing addition of nitrite. The relative rates of production of C 1, C 2, and C 3 alkyl nitrates were roughly constant in all production experiments, and similar to the concentration ratios in ambient waters. This constancy most likely reflects consistency in the ratios of the various organic peroxy radicals involved in the reaction ROO + NO. Alkyl nitrate production rates at 1 μM nitrite concentrations generally did not vary markedly by water type, indicating that distribution of nitrite, not organic matter may control photochemical alkyl nitrate formation in the oceans. At higher nitrite levels, differences in production rates may reflect variations in the concentration or reactivity of dissolved organics.

A potential tolerogenic immune mechanism in a trophoblast cell line through the activation of chemokine-induced T cell death and regulatory T cell modulation

Successful implantation is followed by a local pro-inflammatory and Th1 response, subsequently controlled by Th2. Regulated upon activation, normal T cell expressed and secreted (RANTES) promotes a Th1 response and is implicated as a physiologic tolerogenic factor; therefore, we studied its potential role in the trophoblast-maternal leukocyte dialog. We performed co-cultures of immortalized trophoblast cell line (Swan 71) and peripheral blood mononuclear cells (PBMCs) from fertile women (n = 23) or with recurrent spontaneous abortions (n = 18, RSA). After 24 and 48 h, supernatant and cells were analyzed by enzyme-linked immunosorbent assay, fluorescence-activated cell sorting, Western blot and apoptosis assay. To investigate the physiological effects at peripheral level, we co-cultured maternal and paternal PBMCs with conditioned media from Swan cells and progesterone. Following interaction of maternal PBMCs and trophoblast cells, RANTES production increased (P < 0.05) and was accompanied by low levels of interferon gamma, interleukin-12 p70 and high levels of tumor necrosis factor-alpha, nitrites and leukemia-inhibitory factor. RANTES production resulted in elevated apoptosis of potentially deleterious maternal CD3+ lymphocytes, accompanied by a decrease in the proliferative maternal response. During fetal-maternal dialog, the anti-RANTES antibody significantly reduced the frequency of CD4+CD25+Foxp3+ cells (P < 0.05) and was associated with trophoblast cell survival. However, co-cultures of Swan cells and RSA-PBMCs displayed a differential RANTES kinetics, lower levels of regulatory T cells (Tregs) and CD3+annexin-V+cells, accompanied by higher levels of apoptotic trophoblast cells. RANTES promotes an adequate pro-implantatory microenvironment that influences trophoblast cell survival and modulates the balance of maternal Treg/T effector lymphocytes in favor of maternal tolerance.

Evaluation of Nitrite in Ready-Made Soups

As time is very important for today’s people, ready-made soups in different forms (powder, liquid, canned) are consumed highly. Nitrite has been implicated with a variety of long-term adverse effects and has been of interest to public health providers and governmental regulators for the last 40 years. The present study was aimed to assess nitrite levels in commercially available soups in powder form by using a spectrophotometric method and to evaluate the possible toxicological outcomes. For this purpose, 66 ready-made soups were randomly collected and divided into seven groups: tomato-, chicken-, yoghurt-, mushroom-, lentil-, meat-, and vegetable-based. The minimum–maximum and median levels of nitrite content of the soup groups are 28–2,091 (median, 108), 53–400 (median, 136), 26–129 (median, 47), 58–197 (median, 109), 12–225 (median, 38), 12–555 (median, 40), 184–1229 (median, 389) mg kg−1, respectively. Meat- and tomato-based soups have the highest nitrite contamination among the groups. On the other hand, acceptable daily intake (ADI) of nitrite given by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) and Scientific Committee on Food (SCF) is 0–0.07 mg kg−1 (body weight, b.w.) per day, while Environmental Protection Agency (EPA) has set a reference dose (RfD) of 0.10 mg nitrite nitrogen per kilogram b.w. per day (equivalent to 0.33 mg nitrite ion per kilogram b.w. per day). This means that the highest intake of nitrite a 70-kg person must be no more than 4.9 mg in 1 day according to JECFA and SCF and 7 mg kg−1 b.w. per day according to EPA. However, nitrite intake from one portion (13 g) meat-based soups, when calculated with median daily intake levels (5.05 mg), exceeds the limit of JECFA and SCF. With long-term daily consumption of high nitrite levels exceeding ADI, the risk of mild to moderate methemoglobinemia would be increased, especially for susceptible populations such as young children and elderly. Besides, the higher intake of nitrite from foodstuff and other sources may induce the formation of carcinogenic nitrosamines which are formed endogenously from nitrites and nitrates.

Increased levels of urinary nitrite and nitrotyrosine in Yusho victims 40 years after accidental poisoning with polychlorinated biphenyls in Nagasaki, Japan

Forty years have passed since the accidental poisoning with polychlorinated biphenyls (PCB) in Japan in 1968, named Yusho. High concentrations of PCB are still detected in the serum of the Yusho victims. PCB produces superoxide (O(2) (-)) in the metabolic process and we reported high concentrations of serum nitrite, a stable metabolite reflecting nitric oxide (NO), in the Yusho victims. NO reacts with O(2) (-) and immediately produces peroxynitrite (ONOO(-)). ONOO(-) causes nitration of tyrosine residues and produces nitrotyrosine (NT). Therefore, we measured urinary concentrations of nitrite and NT in the victims and age-matched controls. The mean urinary concentrations of nitrite and NT were significantly higher than in the controls. There was a positive correlation between urinary nitrite and NT in the Yusho victims. Furthermore, there was a positive correlation between the ratio of urinary NT to nitrite and serum PCB concentrations in the Yusho victims. It was considered that the emergence of some ailments could be presumed to have been caused by high levels of urinary nitrite and NT in the Yusho victims.

Expression and characterization of recombinant interferon gamma (IFN-γ) from the nine-banded armadillo (Dasypus novemcinctus) and its effect on Mycobacterium leprae-infected macrophages

Armadillos (Dasypus novemcinctus) manifest the full histopathological spectrum of leprosy, and are hosts of choice for in vivo propagation of Mycobacterium leprae. Though potentially useful as a model of leprosy pathogenesis, few armadillo-specific reagents exist. We have identified a region of high homology to the interferon gamma (IFN-γ) of other mammals within the recently published armadillo whole genomic sequence. cDNA was made from ConA-stimulated armadillo peripheral blood mononuclear cells (PBMC), amplified, and cloned into a pET expression vector for transformation and over-expression in Escherichia coli. The recombinant protein (rDnIFN-γ) was characterized by western blot and its biological function confirmed with bioassays including intracellular killing of Toxoplasma gondii and induction of indoleamine 2, 3-dioxygenase activity. In using rIFN-γ to activate macrophages from mice, humans or armadillos, similar to humans, rIFN-γ-activated armadillo MΦ did not produce nitrite and or inhibit the viability of M. leprae in vitro. Conversely, murine rIFN-γ-activated mouse MΦ produced high levels of nitrite and killed intracellular M. leprae in vitro. These data indicate that the response of armadillo MΦ to rDnIFN-γ is similar to that which occurs in humans, and demonstrates a potentially important value of the armadillo as a model in leprosy research.

Nitrogen budget for a low-salinity, zero-water exchange culture system: II. Evaluation of isonitrogenous feeding of various dietary protein levels to Litopenaeus vannamei (Boone)

This study evaluated the effects of isonitrogenous feeding (60 g dietary protein per kilogram of body weight per day) using experimental feeds with 25%, 30%, 35% and 40% protein on the nitrogen budget, ammonia efflux rate, growth and survival of juvenile Litopenaeus vannamei raised in a low-salinity (4 g L−1) zero-water exchange culture system for 4 weeks. No significant differences in weight gain or instantaneous growth rate were observed between the dietary treatments with 35% and 40% protein after 3 weeks of study, or between treatments with 25% and 30% protein after 4 weeks of study. High mortality rates were observed for the 35% and 40% protein treatments, probably associated with high nitrite levels (4.80 and 7.36 mg NO2-NL−1 respectively) in water. Among the various dietary treatments, 39–46.3% of feed nitrogen was converted to shrimp biomass, 32.8–38.0% and 14.4–39.9% remained within the system as organic and inorganic nitrogen, respectively, and 32.5–39.3% was unaccounted for. The results of the present study showed high nitrogen utilization efficiencies. However, as the nitrogen loading of the zero-water exchange system increased, so did the nitrogen excretion of shrimp, causing a deteriorated general condition of the shrimp, demonstrated by the low ammonia efflux rates recorded at the end of the trial. This study confirms that low-salinity closed systems are particularly susceptible to nitrogen loading. Thus, in these culture systems, low-protein feeds may perform better as they provide more carbon for heterotrophic bacteria and less nitrogen to be degraded and transformed into nitrogenous wastes.

Age-dependent endothelial dysfunction is associated with failure to increase plasma nitrite in response to exercise

Age-dependent alterations of the vessel wall may predispose older individuals to increased cardiovascular pathology. Aging is associated with an impaired bioactivity of nitric oxide (NO). Plasma nitrite reflects NO-synthase activity under fasting conditions and is an important storage pool of NO. To test the hypothesis that aging is associated with an impaired capacity of the vasculature to increase plasma nitrite during exercise, 29 young and 28 old healthy individuals (25 +/- 1 years and 58 +/- 2 years; P < 0.001) without major cardiovascular risk factors were enrolled. Exercise stress was similar in both groups. Baseline nitrite did not differ (107 +/- 8 vs. 82 +/- 10 nmol/l, young vs. old; n.s.) although a trend toward higher nitrite levels in young individuals was seen. In young subjects, exercise increased plasma nitrite by 38 +/- 7% (P < 0.001) compared to only 13 +/- 8% (P = n.s.) in older subjects. L-NMMA blocked increases of nitrite. Endothelial function, as defined by flow-mediated-dilation (FMD) of the brachial artery via ultrasound, was impaired in older subjects (5.4 +/- 0.4% vs. 6.7 +/- 0.3%; P < 0.01). Multivariate analysis showed that age (P = 0.007), BMI (P = 0.010), and LDL (P = 0.021) were independent predictors of nitrite increase. The fact that aging is associated with an impaired capacity of the vasculature to adequately increase nitrite to physiological stimuli may contribute to attenuated maintenance and further deterioration of vascular homeostasis with aging.

NsrR: a key regulator circumventing Salmonella enterica serovar Typhimurium oxidative and nitrosative stress in vitro and in IFN-γ-stimulated J774.2 macrophages

Over the past decade, the flavohaemoglobin Hmp has emerged as the most significant nitric oxide (NO)-detoxifying protein in many diverse micro-organisms, particularly pathogenic bacteria. Its expression in enterobacteria is dramatically increased on exposure to NO and other agents of nitrosative stress as a result of transcriptional regulation of hmp gene expression, mediated by (at least) four regulators. One such regulator, NsrR, has recently been shown to be responsible for repression of hmp transcription in the absence of NO in Escherichia coli and Salmonella, but the roles of other members of this regulon in Salmonella, particularly in surviving nitrosative stresses in vitro and in vivo, have not been elucidated. This paper demonstrates that an nsrR mutant of Salmonella enterica Serovar Typhimurium expresses high levels of Hmp both aerobically and anaerobically, exceeding those that can be elicited in vitro by supplementing media with S-nitrosoglutathione (GSNO). Elevated transcription of ytfE, ygbA, hcp and hcp is also observed, but no evidence was obtained for tehAB upregulation. The hyper-resistance to GSNO of an nsrR mutant is attributable solely to Hmp, since an nsrR hmp double mutant has a wild-type phenotype. However, overexpression of NsrR-regulated genes other than hmp confers some resistance of respiratory oxygen consumption to NO. The ability to enhance, by mutating NsrR, Hmp levels without recourse to exposure to nitrosative stress was used to test the hypothesis that control of Hmp levels is required to avoid oxidative stress, Hmp being a potent generator of superoxide. Within IFN-γ-stimulated J774.2 macrophages, in which high levels of nitrite accumulated (indicative of NO production) an hmp mutant was severely compromised in survival. Surprisingly, under these conditions, an nsrR mutant (as well as an nsrR hmp double mutant) was also disadvantaged relative to the wild-type bacteria, attributable to the combined oxidative effect of the macrophage oxidative burst and Hmp-generated superoxide. This explanation is supported by the sensitivity in vitro of an nsrR mutant to superoxide and peroxide. Fur has recently been confirmed as a weak repressor of hmp transcription, and a fur mutant was also compromised for survival within macrophages even in the absence of elevated NO levels in non-stimulated macrophages. The results indicate the critical role of Hmp in protection of Salmonella from nitrosative stress within and outside macrophages, but also the key role of transcriptional regulation in tuning Hmp levels to prevent exacerbation of the oxidative stress encountered in macrophages.