Important Reactive Nitrogen Research Articles

A coumarin-naphthalimide-based ratiometric fluorescent probe for nitroxyl (HNO) based on an ICT-FRET mechanism

Nitroxyl (HNO) is an important reactive nitrogen that is associated with various states in physiology and pathology and plays a unique function in living systems. So, it is important to exploit fluorescent probes with high sensitivity and selectivity for sensing HNO. In this paper, a novel ratiometric fluorescent probe for HNO was developed utilizing intramolecular charge transfer (ICT) and fluorescence resonance energy transfer (FRET) mechanisms. The probe selected coumarin as energy donor, naphthalimide as energy receptor and 2-(diphenylphosphino)benzoate as the sensing site for detecting HNO. When HNO was not present, the 2-(diphenylphosphino)benzoate unit of the probe restricted electron transfer and the ICT process could not occur, leading to the inhibition of FRET process as well. Thus, in the absence of HNO the probe displayed the intrinsic blue fluorescence of coumarin. When HNO was added, the HNO reacted with the 2-(diphenylphosphino)benzoate unit of the probe to yield a hydroxyl group which resulting in the opening of ICT process and the occurring of FRET process. Thus, after providing HNO the probe displayed yellow fluorescence. In addition, the probe showed good linearity in the ratio of fluorescence intensity at 545 nm and 472 nm (I545 nm/I472 nm) with a concentration of HNO (0.1–20 μM). The probe processed a detection limit of 0.014 μM and a response time of 4 min. The probe also specifically identified HNO over a wide pH scope (pH = 4.00–10.00), including physiological conditions. Cellular experiments had shown that this fluorescent probe was virtually non-cytotoxic and could be applied for ratiometric sensing of HNO in A549 cells.

Recent progress in organelle-targeting fluorescent probes for the detection of peroxynitrite.

Peroxynitrite (ONOO-), as an important reactive nitrogen species, plays a pivotal role in the regulation of intracellular redox homeostasis, signal transduction, cell growth and metabolism, and other physiological processes. Organelles are important for regulating ONOO-, and the dysregulation of ONOO- in organelles is closely related to various diseases. Therefore, it is essential to monitor ONOO- in cellular organelles, including mitochondria, lysosome, endoplasmic reticulum (ER), Golgi apparatus, and lipid droplets. However, the latest advances in organelle-targeting ONOO- fluorescent probes have not been reviewed systematically. In this review, we focus on the design, sensing mechanism, and organelle-targeting imaging applications of ONOO- fluorescent probes that were reported since 2018. This review will help to facilitate the comprehension of organelle-targeting fluorescent probes for the detection of ONOO-.

Seasonal Hypoxia Enhances Benthic Nitrogen Fixation and Shapes Specific Diazotrophic Community in the Eutrophic Marine Ranch

Recently, a growing number of studies have confirmed that biological nitrogen fixation is also an important reactive nitrogen source in coastal regions. However, how benthic nitrogen fixation and diazotrophic community in coastal regions respond to seasonal hypoxia remains largely unknown. In this study, we investigated the spatiotemporal pattern of potential nitrogen fixation rate and diazotrophic abundance and community in sediments of a eutrophic marine ranch experiencing summer hypoxia using 15N tracing and high throughput sequencing techniques. The results showed that potential nitrogen fixation rates ranged from 0.013 to 10.199 μmol kg−1 h−1, and were significantly enhanced by summer hypoxia (ANOVA, p < 0.05). However, nifH gene abundance peaked in June. The diazotrophic community was dominated by Geobacteraceae (>60%), followed by Desulfobulbaceae (13.61%). Bottom water oxygen, pH, Chl-a concentration, and sediment NH4+ significantly regulated benthic nitrogen fixation, while the variation of diazotrophic community was explained by sediment TOC, TN, and Fe content (p < 0.05). This study highlighted that hypoxia stimulated benthic nitrogen fixation, which counteracted the nitrogen removal by denitrification and anammox, and could further aggregate eutrophication of the coastal marine ranch. Moreover, the result emphasized the importance of nitrogen fixation in coastal regions for the global N budget.

Pulse radiolysis studies of the reactions of nitrogen dioxide with the vitamin B12 complexes cob(II)alamin and nitrocobalamin

Although now recognized to be an important reactive nitrogen species in biological systems that modifies the structures of proteins, DNA and lipids, there are few studies on the reactivity of NO2, including the reactions between NO2 and transition metal complexes. We report kinetic studies on the reactions of NO2 with two forms of vitamin B12 — cob(II)alamin and nitrocobalamin. UV–visible spectroscopy and HPLC analysis of the product solution show that NO2 cleanly oxidizes the metal center of cob(II)alamin to form nitrocobalamin, with a second-order rate constant of (3.5±0.3)×108M−1s−1 (pH7.0 and 9.0, room temperature, I=0.20M). The stoichiometry of the reaction is 1:1. No reaction is detected by UV–visible spectroscopy and HPLC analysis of the product solution when nitrocobalamin is exposed to up to 2.0molequiv. NO2.

Deposition of reactive nitrogen during the Rocky Mountain Airborne Nitrogen and Sulfur (RoMANS) study

Increases in reactive nitrogen deposition are a growing concern in the U.S. Rocky Mountain west. The Rocky Mountain Airborne Nitrogen and Sulfur (RoMANS) study was designed to improve understanding of the species and pathways that contribute to nitrogen deposition in Rocky Mountain National Park (RMNP). During two 5-week field campaigns in spring and summer of 2006, the largest contributor to reactive nitrogen deposition in RMNP was found to be wet deposition of ammonium (34% spring and summer), followed by wet deposition of nitrate (24% spring, 28% summer). The third and fourth most important reactive nitrogen deposition pathways were found to be wet deposition of organic nitrogen (17%, 12%) and dry deposition of ammonia (14%, 16%), neither of which is routinely measured by air quality/deposition networks operating in the region. Total reactive nitrogen deposition during the spring campaign was determined to be 0.45 kg ha −1 and more than doubled to 0.95 kg ha −1 during the summer campaign.

Production of nitric oxide by peripheral blood mononuclear cells from the Florida manatee, Trichechus manatus latirostris

Florida manatees ( Trichechus manatus latirostris) are exposed to many conditions in their habitat that may adversely impact health and impair immune function in this endangered species. In an effort to increase the current knowledge base regarding the manatee immune system, the production of an important reactive nitrogen intermediate, nitric oxide (NO), by manatee peripheral blood mononuclear cells (PBMC) was investigated. PBMC from healthy captive manatees were stimulated with LPS, IFN-γ, or TNF-α, either alone or in various combinations, with NO production assessed after 24, 48, 72, and 96 h of culture. NO production in response to LPS stimulation was significantly greater after 48, 72, or 96 h of culture compared to NO production after 24 h of culture. A specific inhibitor of inducible nitric oxide synthase (iNOS), l-NIL ( l- N 6-(1-iminoethyl)lysine), significantly decreased NO production by LPS-stimulated manatee PBMC. Manatee specific oligonucleotide primers for iNOS were designed to measure expression of relative amounts of mRNA in LPS-stimulated manatee PBMC from captive manatees. NO production by PBMC from manatees exposed to red tide toxins was analyzed, with significantly greater NO production by both unstimulated and LPS stimulated PBMC from red tide exposed compared with healthy captive or cold-stress manatees. Free-ranging manatees produced significantly lower amounts of nitric oxide compared to either captive or red tide rescued manatees. Results presented in this paper contribute to the current understanding of manatee immune function and represent the first report of nitric oxide production in the immune system of a marine mammal.

Atmospheric Deposition of Reactive Nitrogen on Turf Grassland in Central Japan: Comparison of the Contribution of Wet and Dry Deposition

The atmospheric deposition of reactive nitrogen on turf grassland in Tsukuba, central Japan, was investigated from July 2003 to December 2004. The target components were ammonium, nitrate, and nitrite ions for wet deposition and gaseous ammonia, nitric and nitrous acids, and particulate ammonium, nitrate, and nitrite for dry deposition. Organic nitrogen was also evaluated by subtracting the amount of inorganic nitrogen from total nitrogen. A wet-only sampler and filter holders were used to collect precipitation and the atmospheric components, respectively. An inferential method was applied to calculate the dry deposition velocity of gases and particles, which involved the effects of surface wetness and ammonia volatilization through stomata on the dry deposition velocity. The mean fraction of the monthly wet to total deposition was different among chemical species; 37, 77, and 1% for ammoniacal, nitrate-, and nitrite-nitrogen, respectively. The annual deposition of inorganic nitrogen in 2004 was 47 and 48 mmol m−2 yr−1 for wet and dry deposition, respectively; 51% of atmospheric deposition was contributed by dry deposition. The annual wet deposition in 2004 was 20, 27, and 0.07 mmol m−2 yr−1, and the annual dry deposition in 2004 was 35, 7.4, and 5.4 mmol m−2 yr−1 for ammoniacal, nitrate-, and nitrite-nitrogen, respectively. Ammoniacal nitrogen was the most important reactive nitrogen because of its remarkable contribution to both wet and dry deposition. The median ratio of the organic nitrogen concentration to total nitrogen was 9.8, 17, and 15% for precipitation, gases, and particles, respectively.

Lung toxicity of nitrogen mustard may be mediated by nitric oxide and peroxynitrite in rats

Nitric oxide (NO) has previously been shown to be responsible for nitrogen mustard (NM)-induced tissue toxicity. Excessive amounts of NO are known to be able to produce peroxynitrite, an important reactive nitrogen compound, by reacting with superoxide. Previous studies reported that NO synthase inhibitors are able to prevent NM toxicity. The aim of this study was to evaluate whether peroxynitrite is also responsible for NM-induced lung tissue damage in rats. Wistar rats were divided into four groups. NM was injected intratracheally and was treated with the selective inducible nitric oxide synthase (iNOS) inhibitor aminoguanidine (AG) (intraperitoneal) or the peroxynitrite scavenger ebselen (intragastric). Control animals were exposed to saline only. NM injection caused both oxidative and nitrosative stress, reflected by dramatically increased levels of the lipid peroxidation end product malondialdehyde (MDA), iNOS activation and urine nitrite–nitrate (NOx) values. Histopathological evaluation demonstrated lung damage with NM exposure. AG blocked iNOS activation and decreased urine NOx levels, and resulted in less histopathological changes in the lung. Although the histopathological outcome was found to be similar to AG, ebselen did not change urinary NOx or lung iNOS levels. Furthermore, ebselen was more able than AG to protect against MDA accumulation. In conclusion, the ability of ebselen to prevent against lung damage without blocking NO synthesis suggests that peroxynitrites may have an important role in the pathogenesis of NM toxicity in addition to NO.

Metallothionein Induces Site-specific Cleavages in tRNAPhe

It is known that metallothionein (MT) plays a role in the scavenging of free radicals, which is produced under various stress conditions. MT may function as an antioxidant that protects against oxidative damage of DNA, protein, and lipid induced by superoxide anion, hydrogen peroxide, hydroxyl radical, nitric oxide, and peroxynitrite. This study was undertaken to test the hypothesis that MT also protects from RNA damage induced by peroxynitrite, an important reactive nitrogen species that causes a diversity of pathological processes. A cell-free system was used. RNA damage was detected by the mobility of in electrophoresis. Cleavages on tRNA were not induced by 3-morpholinosydnomine, which produces peroxynitrite directly. MT induced tRNA damage which was site specific.�⨀炢�⨀嬀炢�⨀袎�⨀袎�⨀炣�⨀Ȁ炣�⨀Ⴀ�⨀Ⴀ�⨀��⨀଀��⨀ꂡ�⨀ഀꂡ�⨀ၽ�⨀Ḁၽ�⨀�⨀�⨀ꂞ�⨀ሀꂞ�⨀�⨀؀�⨀ᢟ�⨀ᢟ�⨀ゟ�⨀଀ゟ�⨀䢟�⨀᐀䢟�⨀E�⨀ࠀE�⨀郋�⨀Ԁ郋�⨀꣋�⨀܀꣋�⨀梥�⨀ࠀ梥�⨀炥�⨀᐀炥�⨀㢦�⨀ጀ㢦�⨀��⨀ഀ��⨀ꂤ�⨀Ȁꂤ�⨀碧�⨀Ȁ碧�⨀ꃉ�⨀Ȁꃉ�⨀꣉�⨀Ȁ꣉�⨀냉�⨀Ā냉�⨀룉�⨀Ȁ룉�⨀색�⨀Ȁ색�⨀죉�⨀Ā죉�⨀탉�⨀Ā탉�⨀꣇�⨀ᜀ꣇�⨀��⨀Ā��⨀�⨀Ā�⨀䄇돀�⨀堘�⨀⁾�⨀ᤇ돐

Involvement of the mitogen-activated protein kinase cascade in peroxynitrite-mediated arachidonic acid release in vascular smooth muscle cells.

Eicosanoid production is reduced when the nitric oxide (NO.) pathway is inhibited or when the inducible NO synthase gene is deleted, indicating that the NO. and arachidonic acid pathways are linked. We hypothesized that peroxynitrite, formed by the reaction of NO. and superoxide anion, may cause signaling events leading to arachidonic acid release and subsequent eicosanoid generation. Western blot analysis of rat arterial smooth muscle cells demonstrated that peroxynitrite (100-500 microM) and 3-morpholinosydnonimine (SIN-1; 200 microM) stimulate phosphorylation of extracellular signal-regulated kinase (ERK), p38, and cytosolic phospholipase A(2) (cPLA(2)). We found that peroxynitrite-induced arachidonic acid release was completely abrogated by the mitogen-activated protein/ERK kinase (MEK) inhibitor U0126 and by calcium chelators. With the p38 inhibitor SB-20219, we demonstrated that peroxynitrite-induced p38 phosphorylation led to minor arachidonic acid release, whereas U0126 completely blocked p38 phosphorylation. Addition of arachidonic acid caused p38 phosphorylation, suggesting that arachidonic acid or its metabolites are responsible for p38 activation. KN-93, a specific inhibitor of Ca(2+)/calmodulin-dependent kinase II (CaMKII), revealed no role for this kinase in peroxynitrite-induced arachidonic acid release in our cell system. Together, these results show that in response to peroxynitrite the cell initiates the MEK/ERK cascade leading to cPLA(2) activation and arachidonic acid release. Thus studies investigating the role of the NO. pathway on eicosanoid production must consider the contribution of signaling pathways initiated by reactive nitrogen species. These findings may provide evidence for a new role of peroxynitrite as an important reactive nitrogen species in vascular disease.

Metallothionein Inhibits Peroxynitrite-induced DNA and Lipoprotein Damage

Previous studies have demonstrated that metallothionein functions as an antioxidant that protects against oxidative DNA, protein, and lipid damage induced by superoxide anion, hydrogen peroxide, hydroxyl radical, and nitric oxide. The present study was undertaken to test the hypothesis that metallothionein also protects from DNA and lipoprotein damage induced by peroxynitrite, an important reactive nitrogen species that causes a diversity of pathological processes. A cell-free system was used. DNA damage was detected by the mobility of plasmid DNA in electrophoresis. Oxidation of low density lipoprotein was measured by a thiobarbituric acid-reactive substance, which was confirmed by lipid hydroperoxide assay. Plasmid DNA damage and low density lipoprotein oxidation were induced by 3-morpholinosydnomine, which produces peroxynitrite through the reaction between nitric oxide and superoxide anion or by synthesized peroxynitrite directly. DNA damage by 3-morpholinosydnomine was prevented by both metallothionein and superoxide dismutase, whereas the damage caused by peroxynitrite was prevented by metallothionein only. The oxidation of low density lipoprotein by 3-morpholinosydnomine and peroxynitrite was also significantly inhibited by metallothionein. This study thus demonstrates that metallothionein may react directly with peroxynitrite to prevent DNA and lipoprotein damage induced by this pathological reactive nitrogen species.

Reactive nitrogen and ozone over the western Pacific: Distribution, partitioning, and sources

Measurements of important reactive nitrogen species (NO, NO2, HNO3, PAN, PPN, NO3−, NOy), C1 to C6 hydrocarbons, O3, chemical tracers (C2Cl4, CO), and meteorological parameters were made in the troposphere (0 to 12 km) over the western Pacific (0°–50°N) during the Pacific Exploratory Mission‐West A campaign (September–October 1991). Under clean conditions, mixing ratios of NO, NO2, NOy, and O3 increased with altitude and showed a distinct latitudinal gradient. PAN showed a midtropospheric maximum, while nitric acid mixing ratios were generally highest near the surface. Measured NOy concentrations were significantly greater than the sum of individually measured nitrogen species (mainly NOx, PAN, and HNO3), suggesting that a large fraction of reactive nitrogen present in the atmosphere is made up of hitherto unknown species. This shortfall was larger in the tropics (≈65%) compared to midlatitudes (≈40%) and was minimal in air masses with high HNO3 mixing ratios (>100 ppt). A global three‐dimensional photochemical model has been used to compare observations with predictions and to assess the significance of major sources. It is possible that the tropical lightning source is much greater than commonly assumed, and both lightning source and its distribution remain a major area of uncertainty in the budgets of NOy and NOx. A large disagreement between measurement and theory exists in the atmospheric distribution of HNO3. It appears that surface‐based anthropogenic emissions provide nearly 65% of the global atmospheric NOy reservoir. Relatively constant NOx/NOy ratios imply that NOy and NOx are in chemical equilibrium and the NOy reservoir may be an important in situ source of atmospheric NOx. Data are interpreted to suggest that only about 20% of the upper tropospheric (7–12 km) NOx is directly attributable to its surface NOx source, and free tropospheric sources are dominant. In situ release of NOx from the NOy reservoir, lightning, direct transport of surface NOx, aircraft emissions, and small stratospheric input collectively maintain the NOx balance in the atmosphere. It is shown that atmospheric ratios of reactive nitrogen and sulfur species, along with trajectory analysis, can be used to pinpoint the source of Asian continental outflow. Compared to rural atmospheres over North America, air masses over the Pacific are highly efficient in net O3 production. Sources of tropospheric NOx cannot yet be accurately defined due to shortcomings in measurements and theory.

Large‐scale distribution of peroxyacetylnitrate from aircraft measurements during the TROPOZ II experiment

Aircraft measurements of peroxyacetylnitrate (PAN) were performed during the TROPOZ II meridional campaign. A specially designed automatic PAN analyzer on board the airplane allowed us to obtain 422 PAN measurements. The data set covers a latitude range from 60 °N to 60 °S and altitudes up to 11 km. PAN was found to be an important reactive nitrogen species with a mixing ratio range of 4 part per trillion (ppt) (percentile 5)–1060 ppt (percentile 95). PAN mixing ratio showed a high degree of variability in both hemispheres. In the southern hemisphere, the higher troposphere exhibited a mixing ratio generally below 20 ppt. The highest mixing ratios were observed in the boundary layer. In polluted areas, PAN levels, higher than 100 ppt, were measured in the lower layers (0–5 km above sea level). In the northern hemisphere, concentrations of PAN ranged from 100–250 ppt in the lower troposphere to 20–40 ppt above 5 km. As a matter of fact, we have frequently measured high PAN mixing ratio (> 200 ppt) over West Africa. Since very few anthropogenic PAN precursors are normally released in this area, these high PAN levels were probably due to bush fires.

Atmospheric measurements of peroxyacetyl nitrate and other organic nitrates at high latitudes: Possible sources and sinks

Aircraft measurements of peroxyacetyl nitrate (PAN) and other important reactive nitrogen species (NO, NO2, HNO3, peroxypropionyl nitrate (PPN), CH3ONO2, NOy) were performed at high latitudes over North America and Greenland during July–August 1988, at all altitudes between 0 and 6 km as part of an Arctic Boundary Layer Expedition (ABLE 3A). Complementing these were measurements of C1 to C5 hydrocarbons, O3, chemical tracers (C2Cl4, CO), and important meteorological parameters. PAN was found to be an important reactive nitrogen species in the free troposphere, with 95% of the mixing ratios falling in the range of 5 to 450 ppt. PAN increased systematically with height with mixing ratios of 100–700 ppt at 6 km and 0–50 ppt in the boundary layer. The free tropospheric PAN reservoir was present over the entire high‐latitude region sampled (50° to 82°N latitude and 60° to 160°W longitude). In the boundary layer, PAN mixing ratios were higher over land than over the North Pacific Ocean. Significant levels of PAN were measured within stratospheric intrusions, forest fire plumes, and episodes of remote pollution. Other organic nitrates such as PPN and CH3ONO2 were found to be a small fraction of PAN (0–10%). PAN and O3 were strongly correlated both in their fine and gross structures, and the latitudinal distribution of PAN in the free troposphere followed that of O3. A two dimensional global photochemical model is used to compare measurements and model results. Model simulations, correlations between reactive nitrogen species (e.g. PAN and NOy) and anthropogenic tracers (C2H2, CO, C2Cl4), and the composition of NOy itself support the view that the reactive nitrogen measured during ABLE 3 A is predominantly of anthropogenic origin with a minor stratospheric component. Transported industrial pollution, biomass burning, and the unique seasonal dynamics of the Arctic/sub‐Arctic region play a dominant role in defining this reactive nitrogen abundance. This PAN (and NOy) reservoir may contribute to the summertime maximum in deposited nitrate observed over Greenland.

Peroxyacetyl nitrate measurements during CITE 2: Atmospheric distribution and precursor relationships

Aircraft measurements of peroxyacetyl nitrate (PAN) and other important reactive nitrogen species (NO, NO2, HNO3, and NOy) were performed over the continental United States and the eastern Pacific during August–September 1986 at all altitudes between O and 6 km as part of CITE 2. PAN measurements were conducted by two independent groups, allowing both intercomparisons and greater confidence in its observed atmospheric structure. PAN was found to be a dominant reactive nitrogen species in the troposphere with 98% of the mixing ratios falling in a range of 5–400 ppt. Typically, the highest mixing ratios (100–300 ppt) were observed aloft (4–6 km) with extremely low values (5–20 ppt) in the marine boundary layer. In the lower troposphere, continental air contained significantly more PAN than marine air. The vertical structure of PAN was largely dictated by its thermal destruction rate and equilibrium with available NO2. PAN mixing ratios showed a high degree of variability in both continental and marine atmospheres. Westerly marine air trajectories did not guarantee well‐mixed air of uniform composition. Mixing ratios of O3, NOy, NOx, HNO3, C2H6, CO, and CFCl3 were strongly correlated with those of PAN, indicating the important role played by transport processes. High PAN to NOx ratios in the mid‐troposphere further support the importance of long‐range transport from continental sources. Frequently, descending air masses from the upper troposphere suggested that PAN mixing ratios probably continued to increase above the 6‐km ceiling altitude. Air masses with O3 <20 ppb, CO <60 ppb, and C2H6 <500 ppt contained only miniscule amounts of PAN and are expected to be of tropical origin. Reasons for the observed PAN variability are discussed.