Nitrosating Agent Research Articles

The Use of Potassium/Sodium Nitrite as a Nitrosating Agent in the Electrooxidative N‐Nitrosation of Secondary Amines

AbstractWe report herein on the electrochemical N‐nitrosation of secondary amines using widely available sodium/potassium nitrite as a nitrosating agent. This approach not only eliminates the need for using a combination of sodium/potassium and a strong acid but also has good functional group tolerance. The reaction is compatible with the late‐stage modification of pharmaceutical compounds and could be conducted in gram scale with a high reaction efficiency. Preliminary mechanistic studies indicate that the N‐nitrosation occurs via the anodic oxidation of KNO2 into an NO2 radical which is then transformed into an NO+ cation.

Nitrosamine Contamination in Pharmaceuticals: Threat, Impact, and Control.

Nitrosamine-contaminated medicinal products have raised safety concerns towards the use of various drugs, not only valsartan and all tetrazole-containing angiotensin II receptor blockers, but also ranitidine, metformin, and other medicines, many of which have been recalled and prone to shortage. At any stages, from drug substance synthesis throughout each product's lifetime, these impurities may evolve if an amine reacts with a nitrosating agent coexisting under appropriate conditions. Consequently, drug regulatory authorities worldwide have established stringent guidelines on nitrosamine contamination for all drug products in the market. This review encompasses various critical elements contributing to successful control measures against current and upcoming nitrosamine issues, ranging from accumulated knowledge of their toxicity concerns and potential root causes, precise risk evaluation, as well as suitable analytical techniques with sufficient sensitivity for impurity determination. With all these tools equipped, the impact of nitrosamine contamination in pharmaceuticals should be mitigated. An evaluation aid to tackle challenges in risk identification, as well as suitable industry-friendly analytical techniques to determine nitrosamines and other mutagenic impurities, are among unmet needs that will significantly simplify the risk assessment process.

Constitutive activation of nitrate reductase in tobacco alters flowering time and plant biomass

Pyridine alkaloids produced in tobacco can react with nitrosating agents such as nitrite to form tobacco-specific nitrosamines (TSNA), which are among the most notable toxicants present in tobacco smoke. The market type known as burley tobacco is particularly susceptible to TSNA formation because its corresponding cultivars exhibit a nitrogen-use-deficiency phenotype which results in high accumulation of nitrate, which, in turn, is converted to nitrite by leaf surface microbes. We have previously shown that expression of a constitutively activated nitrate reductase (NR) enzyme dramatically decreases leaf nitrate levels in burley tobacco, resulting in substantial TSNA reductions without altering the alkaloid profile. Here, we show that plants expressing a constitutively active NR construct, designated 35S:S523D-NR, display an early-flowering phenotype that is also associated with a substantial reduction in plant biomass. We hypothesized that crossing 35S:S523D-NR tobaccos with burley cultivars that flower later than normal would help mitigate the undesirable early-flowering/reduced-biomass traits while maintaining the desirable low-nitrate/TSNA phenotype. To test this, 35S:S523D-NR plants were crossed with two late-flowering cultivars, NC 775 and NC 645WZ. In both cases, the plant biomass at harvest was restored to levels similar to those in the original cultivar used for transformation while the low-nitrate/TSNA trait was maintained. Interestingly, the mechanism by which yield was restored differed markedly between the two crosses. Biomass restoration in F1 hybrids using NC 645WZ as a parent was associated with delayed flowering, as originally hypothesized. Unexpectedly, however, crosses with NC 775 displayed enhanced biomass despite maintaining the early-flowering trait of the 35S:S523D-NR parent.

The Peroxidatic Thiol of Peroxiredoxin 1 is Nitrosated by Nitrosoglutathione but Coordinates to the Dinitrosyl Iron Complex of Glutathione.

Protein S-nitrosation is an important consequence of NO●·metabolism with implications in physiology and pathology. The mechanisms responsible for S-nitrosation in vivo remain debatable and kinetic data on protein S-nitrosation by different agents are limited. 2-Cys peroxiredoxins, in particular Prx1 and Prx2, were detected as being S-nitrosated in multiple mammalian cells under a variety of conditions. Here, we investigated the kinetics of Prx1 S-nitrosation by nitrosoglutathione (GSNO), a recognized biological nitrosating agent, and by the dinitrosyl-iron complex of glutathione (DNIC-GS; [Fe(NO)2(GS)2]−), a hypothetical nitrosating agent. Kinetics studies following the intrinsic fluorescence of Prx1 and its mutants (C83SC173S and C52S) were complemented by product analysis; all experiments were performed at pH 7.4 and 25 ℃. The results show GSNO-mediated nitrosation of Prx1 peroxidatic residue ( = 15.4 ± 0.4 M−1. s−1) and of Prx1 Cys83 residue ( = 1.7 ± 0.4 M−1. s−1). The reaction of nitrosated Prx1 with GSH was also monitored and provided a second-order rate constant for Prx1Cys52NO denitrosation of = 14.4 ± 0.3 M−1. s−1. In contrast, the reaction of DNIC-GS with Prx1 did not nitrosate the enzyme but formed DNIC-Prx1 complexes. The peroxidatic Prx1 Cys was identified as the residue that more rapidly replaces the GS ligand from DNIC-GS ( = 7.0 ± 0.4 M−1. s−1) to produce DNIC-Prx1 ([Fe(NO)2(GS)(Cys52-Prx1)]−). Altogether, the data showed that in addition to S-nitrosation, the Prx1 peroxidatic residue can replace the GS ligand from DNIC-GS, forming stable DNIC-Prx1, and both modifications disrupt important redox switches.

Mycobacterium tuberculosis WhiB3 maintains redox homeostasis and survival in response to reactive oxygen and nitrogen species

Mycobacterium tuberculosis (Mtb) survives under oxidatively and nitosatively hostile niches inside host phagocytes. In other bacteria, adaptation to these stresses is dependent upon the redox sensitive two component systems (e.g., ArcAB) and transcription factors (e.g., FNR/SoxR). However, these factors are absent in Mtb. Therefore, it is not completely understood how Mtb maintains survival and redox balance in response to reactive oxygen species (ROS) and reactive nitrogen species (RNS). Here, we present evidences that a 4Fe-4S-cofactor containing redox-sensitive transcription factor (WhiB3) is exploited by Mtb to adapt under ROS and RNS stress. We show that MtbΔwhiB3 is acutely sensitive to oxidants and to nitrosative agents. Using a genetic biosensor of cytoplasmic redox state (Mrx1-roGFP2) of Mtb, we show that WhiB3 facilitates recovery from ROS (cumene hydroperoxide and hydrogen peroxide) and RNS (acidified nitrite and peroxynitrite). Also, MtbΔwhiB3 displayed reduced survival inside RAW 264.7 macrophages. Consistent with the role of WhiB3 in modulating host-pathogen interaction, we discovered that WhiB3 coordinates the formation of early human granulomas during interaction of Mtb with human peripheral blood mononuclear cells (PBMCs). Altogether, our study provides empirical proof that WhiB3 is required to mitigate redox stress induced by ROS and RNS, which may be important to activate host/bacterial pathways required for the granuloma development and maintenance.

Examining the effect of common nitrosating agents on chitosan using a glucosamine oligosaccharide model system

Chitosan has received substantial attention as a biomaterial due to its unique properties. It has become increasingly common to derivatize chitosan to produce nitric oxide (NO)-releasing materials that exert various therapeutic effects through the action of NO. It is generally the case that these NO-releasing polymers are prepared by exposure to high-pressure NO or nitrosating agents like nitrous acid (HNO2) or alkyl nitrites (RONO). In our study, mass spectrometry and spectroscopic methods demonstrate that both monomeric and oligomeric glucosamine experience chemical alteration after exposure to HNO2-based nitrosating conditions from the literature. In polymeric chitosan, HNO2-based nitrosating conditions were found to induce degradation through the formation of 2,5-anhydro-d-mannose and oligosaccharides. In contrast, the RONO tert-butyl nitrite and high-pressure NO were not found to significantly degrade or otherwise alter the structure of glucosamine or its oligomers, supporting the suitability of these approaches.

Risk assessment of N-nitrosodiethylamine (NDEA) and N-nitrosodiethanolamine (NDELA) in cosmetics

ABSTRACTN-nitrosamines and their precursors found in cosmetics may be carcinogenic in humans. Thus the aim of this study was to carry out risk assessment for N-nitrosamines (N-nitrosodiethanolamine [NDELA], N-nitrosodiethylamine [NDEA]) and amines (triethanolamine [TEA], diethanolamine [DEA]) levels in cosmetics determined using validated liquid chromatography–tandem mass spectrometry (LC–MS/MS) procedures. NDELA and NDEA concentrations were present at levels of “not detected” (N.D.) to 596.5 μg/kg and N.D. to 40.9 μg/kg, respectively. TEA and DEA concentrations ranged from N.D. to 860 μg/kg and N.D. to 26.22 μg/kg, respectively. The nitrite concentration (3–2250 mg/l), number of nitrosating agents to a maximum 5, and pH (3.93–10.09) were also assessed. The impact of N-nitrosamine formation on the levels of TEA, DEA, nitrite, and other nitrosating agents was also examined. N-nitrosamine concentrations correlated with the number of nitrosating agents and nitrite concentrations. Data demonstrated that higher nitrite concentrations and a greater number of nitrosating agents increased NDELA and NDEA yields. Further, the presence of TEA and DEA exerted a significant influence on N-nitrosamine formation. Risk assessments, including the margin of exposure (MOE) and lifetime cancer risk (LCR) for N-nitrosamines and margin of safety (MOS) for amines, were calculated using product type, use pattern, and concentrations. Exposure to maximum amounts of NDELA and NDEA resulted in MOE > 10,000 (based upon the benchmark dose lower confidence limit 10%) and LCR <1 × 10−5, respectively. In addition, TEA and DEA concentrations in cosmetic samples resulted in MOS values >100. Therefore, no apparent safety concerns were associated with cosmetic products containing NDELA, NDEA, TEA, and DEA in this study. However, since amines and nitrosating agents produce carcinogenic nitrosamines, their use in cosmetics needs to be minimized to levels as low as technically feasible.

Formation and inhibition of N-nitrosodiethanolamine in cosmetics under pH, temperature, and fluorescent, ultraviolet, and visual light

ABSTRACTN-nitrosodiethanolamine (NDELA), a type of nitrosamine, is a possible human carcinogen that may form in cosmetic products. The aim of this study was to examine the formation and inhibition of NDELA through chemical reactions of secondary amines including mono-ethanolamine, di-ethanolamine (DEA), and tri-ethanolamine (TEA), and sodium nitrite (SN) under varying conditions such as pH, temperature, and fluorescent, ultraviolet (UV), and visual light (VIS) using liquid chromatography-mass spectroscopy. In a mixture of TEA and SN under acidic conditions pH 2, residual NDELA concentrations rose significantly under various storage conditions in the following order: 50°C > 40°C > UV (2 W/m2) > VIS (4000 lux) > fluorescent light > 25°C > 10°C. In a mixture of DEA and SN under the same acidic pH 2 conditions, NDELA formation was significantly elevated in the following order: UV (2 W/m2) > VIS (4000 lux) > 50°C > 40°C > fluorescent light > 25°C > 10°C. Inhibition of NDELA formation by d-mannitol, vitamin C (Vit C), or vitamin E (Vit E) was determined under varying conditions of pH, temperature, and fluorescent, UV, and VIS. At high concentrations of 100 or 1000 µg/ml, Vit E significantly decreased residual NDELA compared with control levels under acidic pH 2, but not under basic pH 6. Among various antioxidants, Vit E reacted more effectively with many nitrosating agents such as nitrate and nitrite found in cosmetic products. Therefore, to reduce NDELA, it is recommended that cosmetics be stored under cool/amber conditions and that Vit E or Vit C inhibitors of nitrosation be optimally added to cosmetic formulations at concentrations between 100 and 1000 µg/ml.

Computational investigation of the nitrosation mechanism of piperazine in CO2 capture

Quantum chemistry calculations and kinetic modeling were performed to investigate the nitrosation mechanism and kinetics of diamine piperazine (PZ), an alternative solvent for widely used monoethanolamine in postcombustion CO2 capture (PCCC), by two typical nitrosating agents, NO2− and N2O3, in the presence of CO2. Various PZ species and nitrosating agents formed by the reactions of PZ, NO2−, and N2O3 with CO2 were considered. The results indicated that the reactions of PZ species having NH group with N2O3 contribute the most to the formation of nitrosamines in the absorber unit of PCCC and follow a novel three-step nitrosation mechanism, which is initiated by the formation of a charge-transfer complex. The reactions of all PZ species with NO2− proceed more slowly than the reactions of PZ species with ONOCO2−, formed by the reaction of NO2− with CO2. Therefore, the reactions of PZ species with ONOCO2− contribute more to the formation of nitrosamines in the desorber unit of PCCC. In view of CO2 effect on the nitrosation reaction of PZ, the effect through the reaction of PZ with CO2 shows a completely different tendency for different nitrosating agents. More importantly, CO2 can greatly accelerate the nitrosation reactions of PZ by NO2− through the formation of ONOCO2− in the reaction of CO2 with NO2−. This work can help to better understand the nitrosation mechanism of diamines and in the search for efficient methods to prevent the formation of carcinogenic nitrosamines in CO2 capture unit.

Nitrosamine Formation in Amine-Based CO2 Capture in the Absence of NO2: Molecular Modeling and Experimental Validation.

A computational chemistry approach was used to elucidate and verify the different nitrosamine formation mechanisms and pathways. These included nitrosamine formation under acid or basic environments in the presence of NO, O2, SO2 and CO2 without NO2. The results clearly showed that nitrosamine could be formed without NO2 via 2 different types of mechanisms, namely, addition and elimination forming N-N bond before proton transfer and proton transfer before N-N bond formation, respectively. The essence of these mechanisms identified in this work was that two reaction steps were required to complete both reaction mechanisms with different nitrosating agents. Two steps were both necessary neither of which could be neglected, if the nitrosamine formation reaction was to be completed. Computational simulation performed on the reactant, intermediate, transition state, and product for each set of reactions also validated the proposed mechanisms. Experiment also detected nitrosamine from the reaction of diethylamine and NO, SO2, O2, and CO2 in both liquid and gas phase. Thus, NO2 is not necessary for nitrosamine formation to occur in the CO2 capture system.

Inhibitory nitrosylation of mammalian thioredoxin reductase 1: Molecular characterization and evidence for its functional role in cellular nitroso-redox imbalance

Mammalian thioredoxin 1 (Trx1) and the selenoprotein Trx reductase 1 (TrxR1) are key cellular enzymes that function coordinately in thiol-based redox regulation and signaling. Recent studies have revealed that the Trx1/TrxR1 system has an S-nitrosothiol reductase (denitrosylase) activity through which it can regulate nitric oxide-related cellular processes. In this study we revealed that TrxR1 is itself susceptible to nitrosylation, characterized the underlying mechanism, and explored its functional significance. We found that nitrosothiol or nitric oxide donating agents rapidly and effectively inhibited the activity of recombinant or endogenous TrxR1. In particular, the NADPH-reduced TrxR1 was partially and reversibly inhibited upon exposure to low concentrations (<10μM) of S-nitrosocysteine (CysNO) and markedly and continuously inhibited at higher doses. Concurrently, TrxR1 very efficiently reduced low, but not high, levels of CysNO. Biochemical and mass spectrometric analyses indicated that its active site selenocysteine residue renders TrxR1 highly susceptible to nitrosylation-mediated inhibition, and revealed both thiol and selenol modifications at the two redox active centers of the enzyme. Studies in HeLa cancer cells demonstrated that endogenous TrxR1 is sensitive to nitrosylation-dependent inactivation and pointed to an important role for glutathione in reversing or preventing this process. Notably, depletion of cellular glutathione with l-buthionine-sulfoximine synergized with nitrosating agents in promoting sustained nitrosylation and inactivation of TrxR1, events that were accompanied by significant oxidation of Trx1 and extensive cell death. Collectively, these findings expand our knowledge of the role and regulation of the mammalian Trx system in relation to cellular nitroso-redox imbalance. The observations raise the possibility of exploiting the nitrosylation susceptibility of TrxR1 for killing tumor cells.

Experimental and Theoretical Investigation of Effects of Ethanol and Acetic Acid on Carcinogenic NDMA Formation in Simulated Gastric Fluid.

N-nitrosodimethylamine (NDMA), as a representative of endogenously formed N-nitroso compounds (NOCs), has become the focus of considerable research interest due to its unusually high carcinogenicity. In this study, effects of ethanol and acetic acid on the formation of NDMA from dimethylamine (DMA) and nitrite in simulated gastric fluid (SGF) were investigated. Experimental results showed that ethanol in the concentrations of 1-8% (v/v) and acetic acid in the concentrations of 0.01-8% (v/v) exhibit inhibitory and promotion effects on the formation of NDMA, respectively. Moreover, they are both in a dose-dependent manner with the largest inhibition/promotion rate reaching ∼70%. Further experimental investigations indicate that ethanol and acetic acid are both able to scavenge nitrite in SGF. It implies that there are interactions of ethanol and acetic acid with nitrite or nitrite-related nitrosating agents rather than DMA. Theoretical calculations confirm the above experimental results and demonstrate that ethanol and acetic acid can both react with nitrite-related nitrosating agents to produce ethyl nitrite (EtONO) and acetyl nitrite (AcONO), respectively. Furthermore, the reactivities of ethyl nitrite, acetyl nitrite, and dinitrogen trioxide reacting with DMA were found in the order of AcONO > N2O3 ≫ EtONO. This is probably the main reason why there are completely different effects of ethanol and acetic acid on NDMA formation. On the basis of the above results, two requirements for a potential inhibitor of NOCs formation in SGF were provided. The results obtained in this study will be helpful in better understanding the inhibition/promotion mechanisms of compounds on NDMA formation in SGF and searching for protective substances to prevent carcinogenic NOCs formation.

S-Nitrosation of Conserved Cysteines Modulates Activity and Stability of S-Nitrosoglutathione Reductase (GSNOR).

The free radical nitric oxide (NO(•)) regulates diverse physiological processes from vasodilation in humans to gas exchange in plants. S-Nitrosoglutathione (GSNO) is considered a principal nitroso reservoir due to its chemical stability. GSNO accumulation is attenuated by GSNO reductase (GSNOR), a cysteine-rich cytosolic enzyme. Regulation of protein nitrosation is not well understood since NO(•)-dependent events proceed without discernible changes in GSNOR expression. Because GSNORs contain evolutionarily conserved cysteines that could serve as nitrosation sites, we examined the effects of treating plant (Arabidopsis thaliana), mammalian (human), and yeast (Saccharomyces cerevisiae) GSNORs with nitrosating agents in vitro. Enzyme activity was sensitive to nitroso donors, whereas the reducing agent dithiothreitol (DTT) restored activity, suggesting that catalytic impairment was due to S-nitrosation. Protein nitrosation was confirmed by mass spectrometry, by which mono-, di-, and trinitrosation were observed, and these signals were sensitive to DTT. GSNOR mutants in specific non-zinc-coordinating cysteines were less sensitive to catalytic inhibition by nitroso donors and exhibited reduced nitrosation signals by mass spectrometry. Nitrosation also coincided with decreased tryptophan fluorescence, increased thermal aggregation propensity, and increased polydispersity-properties reflected by differential solvent accessibility of amino acids important for dimerization and the shape of the substrate and coenzyme binding pockets as assessed by hydrogen-deuterium exchange mass spectrometry. Collectively, these data suggest a mechanism for NO(•) signal transduction in which GSNOR nitrosation and inhibition transiently permit GSNO accumulation.

Metal-based optical probes for live cell imaging of nitroxyl (HNO).

Nitroxyl (HNO) is a biological signaling agent that displays distinctive reactivity compared to nitric oxide (NO). As a consequence, these two reactive nitrogen species trigger different physiological responses. Selective detection of HNO over NO has been a challenge for chemists, and several fluorogenic molecular probes have been recently developed with that goal in mind. Common constructs take advantage of the HNO-induced reduction of Cu(II) to Cu(I). The sensing mechanism of such probes relies on the ability of the unpaired electron in a d orbital of the Cu(II) center to quench the fluorescence of a photoemissive ligand by either an electron or energy transfer mechanism. Experimental and theoretical mechanistic studies suggest that proton-coupled electron transfer mediates this process, and careful tuning of the copper coordination environment has led to sensors with optimized selectivity and kinetics. The current optical probes cover the visible and near-infrared regions of the spectrum. This palette of sensors comprises structurally and functionally diverse fluorophores such as coumarin (blue/green emission), boron dipyrromethane (BODIPY, green emission), benzoresorufin (red emission), and dihydroxanthenes (near-infrared emission). Many of these sensors have been successfully applied to detect HNO production in live cells. For example, copper-based optical probes have been used to detect HNO production in live mammalian cells that have been treated with H2S and various nitrosating agents. These studies have established a link between HSNO, the smallest S-nitrosothiol, and HNO. In addition, a near-infrared HNO sensor has been used to perform multicolor/multianalyte microscopy, revealing that exogenously applied HNO elevates the concentration of intracellular mobile zinc. This mobilization of zinc ions is presumably a consequence of nitrosation of cysteine residues in zinc-chelating proteins such as metallothionein. Future challenges for the optical imaging of HNO include devising probes that can detect HNO reversibly, especially because ratiometric imaging can only report equilibrium concentrations when the sensing event is reversible. Another important aspect that needs to be addressed is the creation of probes that can sense HNO in specific subcellular locations. These tools would be useful to identify the organelles in which HNO is produced in mammalian cells and probe the intracellular signaling networks in which this reactive nitrogen species is involved. In addition, near-infrared emitting probes might be applied to detect HNO in thicker specimens, such as acute tissue slices and even live animals, enabling the investigation of the roles of HNO in physiological or pathological conditions in multicellular systems.

Volatile N-nitrosamines in meat products: Potential precursors, influence of processing, and mitigation strategies

ABSTRACTMeat products can be contaminated with carcinogenic N-nitrosamines, which is ascribed to the reaction between a nitrosating agent, originating from nitrite or smoke, and a secondary amine, derived from protein and lipid degradation. Although in model systems it is demonstrated that many amine containing compounds can be converted to N-nitrosamines, the yield is dependent of reaction conditions (e.g., low pH and high temperature). In this article, the influence of the composition of the meat products (e.g., pH, aw, spices) and processing (e.g., ageing, ripening, fermentation, smoking, heat treatment and storage) on the presence and availability of the amine precursors and the N-nitrosamine formation mechanism is discussed. In addition, this article explores the current N-nitrosamine mitigation strategies in order to obtain healthier and more natural meat products.

A Chemical‐Proteomic Platform to Investigate Cysteine S‐Nitrosation

Cysteine residues on proteins play numerous functional roles as catalytic nucleophiles, redox‐active disulfides, or metal‐binding residues. Outside of their direct role in catalysis, cysteines can also regulate protein activity via posttranslational modification by endogenous electrophiles and reactive oxygen species. These modified cysteine residues do not conform to a conserved sequence or structural motif, rendering the global identification of regulatory cysteines a considerable challenge. We hypothesize that cysteines that are hypersensitive to different oxidants or nitrosating agents are likely to be enriched in functional residues that serve to modulate protein activity. To explore this hypothesis, we focused our initial efforts on investigating cysteine susceptibility to nitrosation. S‐nitrosation is a unique posttranslational modification that serves to temporally and spatially control protein activity and localization. In order to identify cysteine residues hypersensitive to nitrosation, we developed a chemical‐proteomic platform that ranks cysteines in the human proteome by sensitivity to a variety of nitric oxide donors. Our proteomic studies identified several known sites of nitrosation, as well as previously unannotated cysteines. We then proceeded to functionally characterize several of the unannotated cysteines using a variety of cell and molecular biology approaches. We show that many of these cysteines are located distal from the active site, and yet mediate essential protein functions such as substrate binding and localization. Overall, our studies aim to unearth novel functional cysteine residues in the proteome, with the long‐term goal of identifying new modes of protein regulation through reactive cysteines.

ChemInform Abstract: AgONO‐Assisted Direct C—H Arylation of Heteroarenes with Anilines.

AbstractThe process includes an in situ diazonium reaction with the use of silver nitrite as an unconventional nitrosating agent under acid‐free conditions.

Action of nitroheterocyclic drugs against Clostridium difficile

The nitroheterocyclic classes of drugs have a long history of use in treating anaerobic infections, as exemplified by metronidazole as a first-line treatment for mild-to-moderate Clostridium difficile infection (CDI). Since direct comparisons of the three major classes of nitroheterocyclic drugs (i.e. nitroimidazole, nitazoxanide and nitrofurans) and nitrosating agents against C. difficile are under-examined, in this study their actions against C. difficile were compared. Results show that whilst transient resistance occurs to metronidazole and nitazoxanide, stable resistance arises to nitrofurans upon serial passage. All compounds killed C. difficile at high concentrations in addition to the host defence nitrosating agent S-nitrosoglutathione (GSNO). This suggests that GSNO killing of C. difficile contributes to its efficacy in murine CDI. Although nitric oxide production could not be detected for the nitroheterocyclic drugs, the cellular response to metronidazole and nitrofurans has some overlap with the response to GSNO, causing significant upregulation of the hybrid-cluster protein Hcp that responds to nitrosative stress. These findings provide new insights into the action of nitroheterocyclic drugs against C. difficile.

The small RNA RyhB homologs from Salmonella typhimurium participate in the response to S-nitrosoglutathione-induced stress

Typically, the expression of sRNAs is activated in response to environmental stimuli in order to regulate gene expression through post-transcriptional mechanisms. In the present work we show that the Salmonellatyphimurium paralogous sRNAs RyhB-1 and RyhB-2 are induced in response to the nitrosating agent S-nitrosoglutathione (GSNO). Inactivation of these sRNAs decreased S. typhimurium resistance to GSNO and increased the levels of nitrosylated proteins. These results prompted us to evaluate a possible role of these sRNAs in nitrosative stress resistance. RNA profiling was used as a screen to identify novel RyhB-1 and RyhB-2 regulated targets. A subset of genes was filtered based on their potential role in the response to nitrosative stress and their expression was analyzed by quantitative RT-PCR in wild type, single and double mutant strains (ΔryhB1, ΔryhB2 and ΔryhB1 ΔryhB2) treated with GSNO. In response to GSNO RyhB-1 and RyhB-2 negatively regulate the expression of the genes cyoABC (cytochrome bo oxidase), cydB (cytochrome bd oxidase), cybC (cytochrome b-562), and positively regulate the nirBCD operon (nitrite reductase system). Together, these results suggest that RyhB-1 and RyhB-2 finely tune the expression of genes coding for cytochrome oxidases and the nitrate reductase system, allowing the cell to cope with GSNO-induced stress.

The mutagenicity analysis of imidapril hydrochloride and its degradant, diketopiperazine derivative, nitrosation mixtures by in vitro Ames test with two strains of Salmonella typhimurium

AimThe evaluation of mutagenic properties of imidapril hydrochloride (IMD) and its degradation impurity, diketopiperazine derivative (DKP), nitrosation mixtures was conducted in order to analyze the carcinogenic risk of IMD long-term treatment in patients. In this study an in vitro Ames test with Salmonella enterica serovar Typhimurium TA 98 and TA 100 strains was used. BackgroundIMD and DKP contain nitrogen atoms, which makes them theoretically vulnerable to in vivo nitrosation with the production of N-nitroso compounds (NOC). NOC, in turn, are known animal mutagens indicating that their endogenous production from nitrosable drugs constitutes a carcinogenic hazard. Materials and methodsPure IMD sample was exposed to forced degradation conditions of increased temperature and dry air in order to achieve a DKP sample. Both samples were then treated with a nitrosating agent and the obtained nitrosation mixtures were subjected to mutagenicity analysis by the Ames test with S. typhimurium TA 98 and TA 100 strains in the presence and absence of metabolic activation system (S9 mix) using a commercial Ames MPF 98/100 microplate format mutagenicity assay kit. ResultsNone of the six concentrations of the investigated nitrosation mixtures exhibited any mutagenic potential in both S. typhimurium strains. The addition of S9 mix did not alter the non-mutagenic properties of the studied compounds. ConclusionsThe nitrite treatment of both studied compounds has no impact on their mutagenic properties under the conditions of the present studies. Hence, IMD and DKP nitrosation mixtures are classified as non-mutagens in this test.