Acid-mediated mutagenicity of tobacco snuff: its possible mechanism

Polar solvent extracts of tobacco snuff under acidic conditions were mutagenic in Salmonella typhimurium. Using the Griess reagent test, nitrite ranging from approximately 1.8 to 5.4 mg/g of snuff was found in the polar fraction of extracts. After acid treatment, nitroso compounds in the amount corresponding to the nitrite concentration were detected. The mutagenic potency of the acid-treated extracts was consistent with the content of nitroso compounds generated. Formation of nitroso compounds and the mutagenic activity under acidic conditions was inhibited by ascorbic acid. The results indicate that a nitrosation process was involved in snuff extracts during acid treatment. Studies related to the source of nitrite in tobacco snuff demonstrated that snuff contained bacteria which were able to reduce nitrate to nitrite and that the amount of nitrite in snuff extracts could be further increased by incubation of the extracts with the bacteria. Since snuff contains a considerable amount of nitrate, it seems that reduction of nitrate in snuff to nitrite by bacteria, and nitrosation of certain constituents in snuff by nitrite under acidic conditions to form mutagenic nitroso compounds are possible mechanisms responsible for the acid-mediated mutagenicity of snuff extracts.     

Nitrite and nitroso compounds can serve as specific catalase inhibitors

Objective: We present evidence that nitrite and nitrosothiols, nitrosoamines and non-heme dinitrosyl iron complexes can reversibly inhibit catalase with equal effectiveness.

Methods: Catalase activity was evaluated by the permanganatometric and calorimetric assays.

Results: This inhibition is not the result of chemical transformations of these compounds to a single inhibitor, as well as it is not the result of NO release from these substances (as NO traps have no effect on the extent of inhibition). It was found that chloride and bromide in concentration above 80 mM and thiocyanate in concentration above 20 μM enhance catalase inhibition by nitrite and the nitroso compounds more than 100 times. The inhibition degree in this case is comparable with that induced by azide.

Discussion: We propose that the direct catalase inhibitor is a positively charged NO-group. This group acquires a positive charge in the active center of enzyme by interaction of nitrite or nitroso compounds with some enzyme groups. Halides and thiocyanate protect the NO⁺ group from hydration and thus increase its inhibition effect. It is probable that a comparatively low chloride concentration in many cells is the main factor to protect catalase from inhibition by nitrite and nitroso compounds.     

Nitrosative stress in an animal model of necrotizing enterocolitis

Necrotizing enterocolitis (NEC) is a disease of newborns characterized by gut barrier failure. We reasoned that upregulation of inducible nitric oxide synthase (iNOS) may result in nitrosative stress and accumulation of nitroso species in the intestine. Newborn rats were either breast-fed (BF), or formula-fed and additionally subjected to hypoxia (FFH). At Day 4 after birth, the distal ilea were harvested and processed for Western blot analysis and measurement of NO-related metabolites. While BF neonates showed normal morphology, FFH neonates developed signs of NEC by Day 4. These pathological changes correlated with upregulation of iNOS and increases in tissue nitrite, nitrosothiol, and nitrosamine concentrations. Enhanced nitroso levels were most prominent in the mucosal layers of the ileum and iNOS inhibition resulted in a significant decrease in both nitroso species and incidence of NEC. In contrast, increased nitrite levels were distributed evenly throughout the ileum and remained unchanged following iNOS inhibition. Similarly, specimens from NEC patients had higher intestinal levels of NO-related metabolites compared to non-NEC controls. This is the first report of tissue levels of nitroso species in the gut of an animal model of NEC and of human specimens. The results suggest that local nitrosative stress contributes to the pathology associated with NEC. Unexpectedly, the NO breakdown product nitrite, previously considered biologically inert, was found to be present throughout the ileal wall, suggesting that cellular NO metabolism is altered significantly in NEC. Whether nitrite plays a protective or deleterious role remains to be investigated.     

Mutagenicity of nitrosated food items.

Several food items, commonly consumed in South India, after nitrite treatment under simulated gastric conditions were found to be mutagenic in Salmonella typhimurium tester strain TA 100. Dichloromethane extracts containing the volatile nitroso compounds and ethyl-acetate extracts with the non-volatile nitroso compounds of some of the food items exhibited mutagenicity.     

Dermal application of nitric oxide releasing acidified nitrite-containing liniments significantly reduces blood pressure in humans

Vascular ischemic diseases, hypertension, and other systemic hemodynamic and vascular disorders may be the result of impaired bioavailability of nitric oxide (NO). NO but also its active derivates like nitrite or nitroso compounds are important effector and signal molecules with vasodilating properties. Our previous findings point to a therapeutical potential of cutaneous administration of NO in the treatment of systemic hemodynamic disorders. Unfortunately, no reliable data are available on the mechanisms, kinetics and biological responses of dermal application of nitric oxide in humans in vivo. The aim of the study was to close this gap and to explore the therapeutical potential of dermal nitric oxide application. We characterized with human skin in vitro and in vivo the capacity of NO, applied in a NO-releasing acidified form of nitrite-containing liniments, to penetrate the epidermis and to influence local as well as systemic hemodynamic parameters. We found that dermal application of NO led to a very rapid and significant transepidermal translocation of NO into the underlying tissue. Depending on the size of treated skin area, this translocation manifests itself through a significant systemic increase of the NO derivates nitrite and nitroso compounds, respectively. In parallel, this translocation was accompanied by an increased systemic vasodilatation and blood flow as well as reduced blood pressure. We here give evidence that in humans dermal application of NO has a therapeutic potential for systemic hemodynamic disorders that might arise from local or systemic insufficient availability of NO or its bio-active NO derivates, respectively.     

Recent methodological advances in the analysis of nitrite in the human circulation: nitrite as a biochemical parameter of the L-arginine/NO pathway

Nitric oxide (NO) plays a pivotal role in the modulation of multiple physiological processes. It acts as a messenger molecule within the cardiovascular system. NO is a highly unstable free radical in circulating blood and is oxidized rapidly to nitrite and nitrate. Recent studies suggest that nitrite has the potential to function as a surrogate of NO production under physiological and pathophysiological conditions and could therefore be of high relevance as a biochemical parameter in experimental and clinical studies. Under hypoxic conditions nitrite is reduced to bioactive NO by deoxyhemoglobin. This mechanism may represent a dynamic cycle of NO generation to adapt the demand and supply for the vascular system. Because of these potential biological functions the concentration of nitrite in blood is thought to be of particular importance. The determination of nitrite in biological matrices represents a considerable analytical challenge. Methodological problems often arise from pre-analytical sample preparation, sample contamination due to the ubiquity of nitrite, and from lack of selectivity and sensitivity. These analytical difficulties may be a plausible explanation for reported highly diverging concentrations of nitrite in the human circulation. The aim of this article is to review the methods of quantitative analysis of nitrite in the human circulation, notably in plasma and blood, and to discuss pre-analytical and analytical factors potentially affecting accurate quantification of nitrite in these human fluids.     

The bacterial mutagenicity of three naturally occurring indoles after reaction with nitrous acid

Three naturally occurring indoles were evaluated for potential nitrosatability using the Nitrosation Assay Procedure (NAP test) as recommended by the World Health Organisation. All three indoles i.e. tryptophan, tryptamine and 5-hydroxy-tryptamine were nitrosated to products which were directly mutagenic for S. typhimurium TA1537. In addition, the products of nitrosation of tryptamine and 5-hydroxytryptamine were also mutagenic for strains TA1538, TA98 and TA1535 without the need for metabolic activation. The sensitivities of the frameshift-detecting strains TA1537, TA1538 and TA98 were of particular interest, since nitroso compounds are characteristically base-substitution mutagens. The mutagenic effects of the products formed after nitrosation of each indole at pH 3.6, were eliminated in the presence of S9 mix. This was not the case when the nitrosation assay was carried out at pH 2.6. At this pH the mutagenicity of the nitrosated products varied in the presence of S9 mix and depended upon the nature of the indole undergoing nitrosation, and the bacterial test strain utilised for the mutagenicity assay. This indicated that more than one mutagenic product was responsible for the observed effects. As well as pH, a number of other factors influenced the formation of mutagenic nitroso products. Most notably, the concentrations of precursor compounds (sodium nitrite, and indole) present in the NAP test were of critical importance. As the sodium nitrite concentration was reduced from that recommended by the W.H.O. (40 mM), so the mutagenicity decreased. For all three compounds significant mutagenic effects were lost at sodium nitrite concentrations below 15 mM. In conclusion the data presented in this paper clearly demonstrates that individuals are chronically exposed to naturally occurring substances which readily nitrosate in excess nitrous acid and yield bacterial mutagens.     

Formation of bacterial mutagens from the reaction of chewing tobacco with nitrite

Using the Salmonella/microsome assay system, the mutagenicity of chewing tobacco extracts (CTE) treated with and without sodium nitrite under acidic conditions was examined. Mutagenic activity was found only for nitrite-treated CTE in both tester strains, TA98 and TA100, and was independent of metabolic activation. Formation of mutagenic substances from CTE by nitrite was dependent on acidic pHs (the highest at pH 2) and could be inhibited by ascorbate. The mutagenic potency of CTE plus nitrite was proportional to the content of nitroso compounds generated in the reaction mixture, indicating that the nitrosation process was involved. The possible in vivo nitrosation and the potential health effect are discussed.     

Dietary nitrite restores NO homeostasis and is cardioprotective in endothelial nitric oxide synthase-deficient mice

Endothelial production of nitric oxide (NO) is critical for vascular homeostasis. Nitrite and nitrate are formed endogenously by the stepwise oxidation of NO and have, for years, been regarded as inactive degradation products. As a result, both anions are routinely used as surrogate markers of NO production, with nitrite as a more sensitive marker. However, both nitrite and nitrate are derived from dietary sources. We sought to determine how exogenous nitrite affects steady-state concentrations of NO metabolites thought to originate from nitric oxide synthase (NOS)-derived NO as well as blood pressure and myocardial ischemia-reperfusion (I/R) injury. Mice deficient in endothelial nitric oxide synthase (eNOS-/-) demonstrated decreased blood and tissue nitrite, nitrate, and nitroso proteins, which were further reduced by low-nitrite (NOx) diet for 1 week. Nitrite supplementation (50 mg/L) in the drinking water for 1 week restored NO homeostasis in eNOS-/- mice and protected against I/R injury. Nitrite failed to alter heart rate or mean arterial blood pressure at the protective dose. These data demonstrate the significant influence of dietary nitrite intake on the maintenance of steady-state NO levels. Dietary nitrite and nitrate may serve as essential nutrients for optimal cardiovascular health and may provide a novel prevention/treatment modality for disease associated with NO insufficiency.     

Nitrite is produced by elicited but not by circulating neutrophils

The generation of nitrite (NO(2) (-)) was used as an index of the production of nitric oxide by human and rat polymorphonuclear leukocytes (PMN) and rat peritoneal macrophages. Human peripheral blood PMN did not produce significant levels of NO(2) (-). Attempts to induce NO(2) (-) generation in human PMN by incubation with GM-CSF (1 nM), TNFalpha (0.3 nM), endotoxin (1 mug/ml) or formyl-Met-Leu-Phe (100 nM) for up to 16 h were not successful. Addition of human PMN primed by GM-CSF (1 nM) to rabbit aortic ring preparations precontracted with phenylephrine had no effect on tone. In contrast to these observations, PMN, isolated from the peritoneum of oyster glycogen treated rats, generated NO(2) (-) via a pathway sensitive to inhibition by the nitric oxide synthase inhibitor, N(G)-monomethyl L-arginine. However, peripheral blood rat PMN obtained from the same animals did not produce NO(2) (-), even during prolonged incubation for periods of up to 16 h. It is suggested that detectable NO production by PMN requires NO synthase activity to be induced either by the process of PMN migration or by exposure to certain cytokines produced locally at the site of inflammation.     

Nitrosation in vitro and in vivo by sodium nitrite, and mutagenicity of nitrogenous pesticides.

37 nitrogenous pesticides, belonging to the chemical groups of amides, carbamates and ureas, were nitrosated with sodium nitrite in vitro. The nitrosated compounds were tested for mutagenic activity in the bacterial spot test with Salmonella typhimurium his G 46. Those pesticides reacting positively in this test after nitrosation were then fed to mice in combination with sodium nitrite in order to assess the formation and mutagenicity of these nitroso compounds in vivo. With the already known exception of ethylenethiourea (ETU), no pesticide produced enhanced numbers of micronuclei in mouse bone-marrow erythrocytes when fed together with nitrite. Dose-response experiments with intraperitoneal injection of N-nitroso-ETU revealed an apparent no-effect level of about 15--18 mg/kg. The findings are correlated with the pesticide residues actually present in the environment.     

Adaptation of the Griess reaction for detection of nitrite in human plasma

The determination of nitrite in human plasma or serum has been most frequently used as a marker of nitric oxide (NO) production. In addition, it has recently been suggested that nitrite could act as a vasodilating agent at physiological concentrations by NO delivery. Therefore, nitrite determination in biological fluids is becoming increasingly important. The most frequently used method to measure nitrite is based on the spectrophotometric analysis of the azo dye obtained after reaction with the Griess reagent. This method has some limitations regarding detection limit and sensitivity, thus resulting unsuitable for nitrite detection in plasma. We have identified some drawbacks and modified the original procedure to overcome these problems. By the use of the newly developed method, we measured 221±72 nM nitrite in human plasma from healthy donors.     

Synthesis and evaluation of some lipidic aminoalcohols and diamines as immunomodulators

Lymphoproliferation inhibition and cytotoxicity of a number of lipidic aminoacids, aminoalcohols and diamines were evaluated as a preliminary screening to select potential immunomodulators. The four most potent/less toxic compounds were submitted to delayed hypersensibility (DTH) assays to define the best to be evaluated further Graft-vs-Host, NO production and other immunoevaluation (CD4(+), CD45, CD8, CD11b, I-Ek, and NK cells) assays, to establish their immunomodulation potential for being further considered as auxiliary agents for vaccination against some parasitic infections. Compounds 5d, 6d, 6f, 7a, and 9a, fairly inhibited the lymphoproliferation (71.6-79.5%, at 3.2-2.4 nM), while the aminoalcohol derivative 6f and the diamine 7a gave the most promising results in the DTH assays. Diamine derivative 8b induced nitrite production on normal macrophages, whereas compounds 6f and 7a induced nitrite production on LPS pre-stimulated macrophages. These two last compounds have been selected to follow in vivo vaccination assays.     

Formation of mutagenic N-nitroso compounds from the pesticides prometryne, dodine and carbaryl in the presence of nitrite at pH 1

Environmental chemicals including pesticides carrying secondary and tertiary amino groups are suggested to be a health hazard to man since potentially carcinogenic nitroso compounds may be formed in the presence of nitrite at low pH values resembling conditions in the human stomach. Nitrosation of the isopropylamino-triazine Prometryne, the n-dodecyl guanidine Dodine and the N-methylcarbamate carbaryl was investigated in the presence of HCl and acetic acid at pH 1 and excess sodium nitrite for 4 h at 37 degrees C. The reaction products were extracted with CCl4 and were analyzed qualitatively and quantitatively by infrared spectroscopy, nuclear-resonance spectrometry, GC/mass spectrometry and by spectrophotometry. All compounds investigated formed N-nitroso derivatives in the following yields: carbaryl 67%, Dodine 12% and Prometryne 14%. The N-nitroso derivatives per se were not or only slightly mutagenic to Escherichia coli K12 or Salmonella typhimurium TA 1538. However, significantly increased mutation frequencies were seen after metabolic activation by mouse-liver microsomes. These results add to the observations that among environmental chemicals not only those containing methyl- or ethyl-substituted amino groups form potentially carcinogenic nitroso derivatives but also those with iso-propylamino groups as well as alkyl-substituted guanidine derivatives.     

Control of artifacts in plasma adenosine determinations

The literature concerning the role of adenosine (ADO) in physiology reveals no agreement about plasma ADO concentrations and suggests two main sources of error in these determinations: rapid ADO uptake by red blood cells or rapid ADO production from ADO nucleotides, which may be released by any cell lysis or platelet aggregation during plasma preparation. We therefore studied ADO concentrations in plasma from normal human forearm venous blood. ADO was determined by a high-performance liquid chromatographic procedure with a sensitivity of 3 nM (original plasma). Observed ADO concentrations ranged from 894 nM to 8.2 nM depending on the conditions of plasma preparation. In plasma prepared in plastic tubes from 4.5 ml of blood drawn into a plastic syringe containing 1.5 ml of an isotonic stopping solution (pH 7.4) containing heparin (60 units ml), dilazep (40 microM), EGTA (40 mM, EDTA (40 mM), erythro-9-(2-hydroxy-3-nonyl) adenine (40 microM), and alpha, beta-methylene adenosine-5'-diphosphate (525 nM), the plasma ADO concentration was 13.3 +/- 1.88 nM (SE) after correction for a simultaneous ADO recovery determination. The mean ADO recovery was 78% +/- 3.39. The mean plasma ADO concentration found by this method of collection and preparation is lower then reported by others. Proper collection methods are required to avoid artifacts when determining plasma ADO concentrations.     

Detection of nitrite and nitrosocompounds in chemical systems and biological liquids by the calorimetric method

The capacity of nitrite, S-nitrosothiols (RS-NO), dinitrosyl iron complexes (DNICs) with thiolcontaining ligands, and nitrosoamines to inhibit catalase has been used for the selective determination of these compounds in purely chemical systems and biological liquids: cow milk and colostrum. The limiting sensitivity of the method is 50 nM. A comparison of the results of the determinations of RS-NO, DNIC, and nitrite by the catalase method and the Griess method conventionally used for nitrite detection showed that, firstly, Griess reagents decompose DNIC and RS-NO to form nitrite. Therefore, the Griess method cannot be used for nitrite determination in solutions of these substances. Secondly, Griess reagents interact with complexes of mercury ions with RS-NO, inducing the release of nitrosonium ions from the complex followed by the hydrolysis of nitrosonium to nitrite. Thus, the proposition about the spontaneous decay of the complexes of mercury ions with RS-NO is incorrect. Keeping in mind a high sensitivity of the method, the use of catalase as an enzyme detector of nitrosocompounds allows one to detect these compounds in neutral medium without prior purification of the object, thereby preventing artificial effects due to noncontrolled modifications of the compounds under study.     

Nitrosation of aspartic acid, aspartame, and glycine ethylester. Alkylation of 4-(p-nitrobenzyl)pyridine (NBP) in vitro and binding to DNA in the rat

In a colorimetric assay using 4-(p-nitrobenzyl)pyridine (NBP) as a nucleophilic scavenger of alkylating agents, the nitrosation and alkylation reactions were investigated for a number of amino acids and derivatives. The alkylating activity increased with the square of the nitrite concentration. The nitrosation rate constants for aspartic acid, aspartame, and glycine ethylester (= precursors C) were 0.08, 1.4 and less than or equal to 0.2, respectively, expressed in terms of the pH-dependent k2 rate constant of the equation dNOC/dt = k2.[C].[nitrite]2. The rates correlated inversely with the basicity of the amino group. The stability of the alkylating activity was astonishingly high, both in acid and at neutral pH. Half-lives of 500, 200, and 30 min were determined for aspartic acid (pH 3.5), aspartame (pH 2.5), and glycine ethylester (pH 2.5). Values of 60, 15, and 2 min, respectively, were found at pH 7. It is concluded that rearrangement of the primary N-nitroso product to the ultimate alkylating agent could be rate-limiting. The potential of nitrosated alpha-amino acids to bind to DNA in vivo was investigated by oral gavage of radiolabelled glycine ethylester to rats, followed immediately by sodium nitrite. DNA was isolated from stomach and liver and analysed for radioactivity and modified nucleotides. No indication of DNA adduct formation was obtained. Based on an estimation of the dose fraction converted from glycine ethylester to the nitroso product under the given experimental conditions, the maximum possible DNA-binding potency of nitroso glycine ethylester is about one order of magnitude below the methylating potency of N-nitrosomethylurea in rat stomach. The apparent discrepancy to the in vitro data could be due to efficient detoxification processes in mammalian cells.     

Measurement of S-nitrosoalbumin by gas chromatography--mass spectrometry. III. Quantitative determination in human plasma after specific conversion of the S-nitroso group to nitrite by cysteine and Cu(2+) via intermediate formation of S-nitrosocysteine and nitric oxide

Highly contradictory data exist on the normal plasma basal levels in humans of S-nitrosoproteins, in particular of S-nitrosoalbumin (SNALB), the most abundant nitric oxide (.NO) transport form in the human circulation with a range of three orders of magnitude (i.e., 10 nM-10 microM). In previous work we reported on a GC-MS method for the quantitative determination of SNALB in human plasma. This method is based on selective extraction of SNALB and its 15N-labeled SNALB analog (S(15)NALB) used as internal standard on HiTrapBlue Sepharose affinity columns, HgCl(2)-catalysed conversion of the S-nitroso groups to nitrite and [15N]nitrite, respectively, their derivatization to the pentafluorobenzyl derivatives and quantification by GC-MS. By this method we had measured SNALB basal plasma levels of 181 nM in healthy humans. It is generally accepted that HgCl(2)-catalysed conversion of S-nitroso groups into nitrite is specific. In consideration of the highly divergent SNALB plasma levels in humans reported so far, we were interested in an additional method that would allow specific conversion of S-nitroso groups into nitrite. We found that treatment with cysteine plus CuSO(4) is as effective and specific as treatment with HgCl(2). The principle of the cysteine/CuSO(4) procedure is based on the transfer of the S-nitroso group from SNALB to cysteine yielding S-nitrosocysteine, and its subsequent highly Cu(2+)-sensitive conversion into nitrite via intermediate.NO formation. Similar SNALB concentrations in the plasma of 10 healthy humans were measured by GC-MS using HgCl(2) (156+/-64 nM) and cysteine/CuSO(4) (205+/-96 nM). Our results strongly suggest that SNALB is an endogenous constituent in human plasma and that its concentration is of the order of 150-200 nM under physiological conditions.     

Stable-isotope dilution GC–MS approach for nitrite quantification in human whole blood,erythrocytes, and plasma using pentafluorobenzyl bromide derivatization: Nitrite distribution in human blood

Previously, we reported on the usefulness of pentafluorobenzyl bromide (PFB-Br) for the simultaneous derivatization and quantitative determination of nitrite and nitrate in various biological fluids by GC–MS using their ¹⁵N-labelled analogues as internal standards. As nitrite may be distributed unevenly in plasma and blood cells, its quantification in whole blood rather than in plasma or serum may be the most appropriate approach to determine nitrite concentration in the circulation. So far, GC–MS methods based on PFB-Br derivatization failed to measure nitrite in whole blood and erythrocytes because of rapid nitrite loss by oxidation and other unknown reactions during derivatization. The present article reports optimized and validated procedures for sample preparation and nitrite derivatization which allow for reliable quantification of nitrite in human whole blood and erythrocytes. Essential measures for stabilizing nitrite in these samples include sample cooling (0–4 °C), hemoglobin (Hb) removal by precipitation with acetone and short derivatization of the Hb-free supernatant (5 min, 50 °C). Potassium ferricyanide (K₃Fe(CN)₆) is useful in preventing Hb-caused nitrite loss, however, this chemical is not absolutely required in the present method. Our results show that accurate GC–MS quantification of nitrite as PFB derivative is feasible virtually in every biological matrix with similar accuracy and precision. In EDTA-anticoagulated venous blood of 10 healthy young volunteers, endogenous nitrite concentration was measured to be 486 ± 280 nM in whole blood, 672 ± 496 nM in plasma (C_P), and 620 ± 350 nM in erythrocytes (C_E). The C_E-to-C_P ratio was 0.993 ± 0.188 indicating almost even distribution of endogenous nitrite between plasma and erythrocytes. By contrast, the major fraction of nitrite added to whole blood remained in plasma. The present GC–MS method is useful to investigate distribution and metabolism of endogenous and exogenous nitrite in blood compartments under basal conditions and during hyperemia.     

Intragastric generation of antimicrobial nitrogen oxides from saliva--physiological and therapeutic considerations

Salivary nitrite is suggested to enhance the antimicrobial properties of gastric juice by conversion to nitric oxide (NO) and other reactive nitrogen intermediates in the stomach. Intubated patients exhibit extremely low gastric levels of NO, because they do not swallow their saliva. The present investigation was designed to examine the antibacterial effects of human saliva and gastric juice. Furthermore, we studied a new mode of NO delivery, involving formation from acidified nitrite, which could prevent bacterial growth in the gastric juice of intubated patients in intensive care units. The growth of Escherichia coli ATCC 25922 and the formation of NO and nitroso/nitrosyl species were determined after incubation of gastric juice with saliva from healthy volunteers that was rich (nitrate ingestion) or poor (overnight fasting) in nitrite. In a stomach model containing gastric juice from intubated patients, we inserted a catheter with a silicone retention cuff filled with ascorbic acid and nitrite and determined the resulting antibacterial effects on E. coli and Candida albicans. Saliva enhanced the bactericidal effect of gastric juice, especially saliva rich in nitrite. Formation of NO and nitroso/nitrosyl species by nitrite-rich saliva was 10-fold greater than that by saliva poor in nitrite. In our stomach model, E. coli and C. albicans were killed after exposure to ascorbic acid and nitrite. In conclusion, saliva rich in nitrite enhances the bactericidal effects of gastric juice, possibly through the generation of reactive nitrogen intermediates, including NO. Acidified nitrite inside a gas-permeable retention cuff may be useful for restoring gastric NO levels and host defense in critically ill patients.