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At least 19 recordsLinked to original sources
PubMed1997

Superoxide modulates the oxidation and nitrosation of thiols by nitric oxide-derived reactive intermediates. Chemical aspects involved in the balance between oxidative and nitrosative stress.

Thiol-containing proteins are key to numerous cellular processes, and their functions can be modified by thiol nitrosation or oxidation. Nitrosation reactions are quenched by O-2, while the oxidation chemistry mediated by peroxynitrite is quenched by excess flux of either NO or O-2. A solution of glutathione (GSH), a model thiol-containing tripeptide, exclusively yielded S-nitrosoglutathione when exposed to the NO donor, Et2NN(O)NONa. However, when xanthine oxidase was added to the same mixture, the yield of S-nitrosoglutathione dramatically decreased as the activity of xanthine oxidase increased, such that there was a 95% reduction in nitrosation when the fluxes of NO and O-2 were nearly equivalent. The presence of superoxide dismutase reversed O-2-mediated inhibition, while catalase had no effect. Increasing the flux of O-2 yielded oxidized glutathione (GSSG), peaking when the flux of NO and O-2 were approximately equivalent. The results suggest that oxidation and nitrosation of thiols by superoxide and NO are determined by their relative fluxes and may have physiological significance.

Free Radicals↗
PubMed1989

The inhibition of bacterially mediated N-nitrosation by vitamin C: relevance to the inhibition of endogenous N-nitrosation in the achlorhydric stomach.

It has been suggested that endogenously formed N-nitroso compounds are involved in the aetiology of gastric cancer. In the model of gastric carcinogenesis postulated by Correa, gastric atrophy is an important early stage in the progression to carcinoma which results in the loss of stomach acidity, and colonization of the stomach by bacteria. As a consequence of the metabolic activity of these bacteria intragastric nitrite (a precursor to N-nitroso compounds) and possibly carcinogenic N-nitroso compounds become elevated, which may hasten the progression to carcinoma. Vitamin C has been shown to be an effective inhibitor of acid-catalysed N-nitroso compound formation, in vivo and in vitro, and this has been attributed to its relatively rapid reaction with nitrite in contrast to the slower rates of reaction of nitrite with secondary amines. However, N-nitroso compound formation in the achlorhydric stomach must proceed by mechanisms which operate at neutral pH values. One potential mechanism involves the enzymatic catalysis of N-nitrosation by a subpopulation of the bacteria colonizing the achlorhydric stomach which catalyse these reactions and in particular denitrifying organisms. In this study, we examined the effect of vitamin C on the formation of N-nitrosomorpholine from morpholine and nitrite when mediated by cells of an actively N-nitrosating denitrifying bacterium (Pseudomonas aeruginosa, BM1030) at neutral pH. Despite the fact that vitamin C ordinarily shows little reactivity towards nitrite at neutral pH it did prove to be a potent inhibitor of bacterial N-nitrosamine formation. This study provides some justification for the use of vitamin C as an inhibitor of endogenous N-nitrosation regardless of gastric pH.

Ascorbic Acid↗
PubMed1988

Mechanism for nitrosation of 2,3-diaminonaphthalene by Escherichia coli: enzymatic production of NO followed by O2-dependent chemical nitrosation.

The mechanism by which Escherichia coli can catalyze the nitrite-dependent nitrosation of 2,3-diaminonaphthalene (DAN), with formation of the corresponding fluorescent triazole, was studied. The reaction was dependent on production of a gaseous compound which can nitrosylate DAN upon contact with air. This compound was identified as nitric oxide (NO), and the kinetics of NO and triazole production are reported. NO and triazole were produced proportionally in a stoichiometric ratio, NO/triazole, of 1.4 to 1.7. Given the requirement for air, nitrosation of DAN probably proceeds via formation of the well-known strong nitrosylating agents N2O3 and N2O4 from NO. The parallel inhibition of NO and triazole production by azide and nitrate served to reinforce the link between nitrosation and nitrate reductase that had been established previously by others on genetic grounds.

2-Naphthylamine↗
PubMed1990

A comparison of the results of sequential hydrazinolysis-nitrosation and alkali-mediated cleavage-nitrosation of the O-linked oligosaccharides of gastric mucus glycoproteins.

Analysis of the oligosaccharides released from pig gastric mucus glycopolypeptides by hydrazinolysis showed that degradation had occurred. Nitrosation of the products followed by reduction gave a mixture that had a low content of 2,5-anhydro-D-talitol, which implied destruction of much of the terminal reducing 2-amino-2-deoxy-D-galactose. Under the conditions of hydrazinolysis, cellobiose was largely unchanged but laminaribiose gave a complex mixture that probably contained glucose hydrazone (13C-n.m.r. data). In order to avoid degradation, the hydrazinolysis-nitrosation sequence should be applied to the reduced oligosaccharides released on cleavage with alkali.

Alkalies↗
PubMed1975

Nitrosation of phenacetin. Formation of N-nitroso-2-nitro-4-ethoxyacetanilide as an unstable product of the nitrosation in dilute aqueous-acidic solution.

Reaction of phenacetin with N2O4 in glacial acetic acid at 10(0) C gives N-nitroso-2-nitro-4-ethoxyacetanilide. This N-nitrosoacylarylamine is stable at low temperatures (--30 degress C) but unstable at ambient temperature. No intact N-nitroso-2-nitro-4-ethoxyacetanilide can be detected when phenacetin is nitrosated under conditions simulating those in the stomach (37 degrees C, pH 1). Instead, 2-nitro-4-ethoxybenzenediazonium chloride is the main reaction product found. Under the conditions applied, the N-nitrosoacylarylamine rapidly rearranges by 1,3-migration of the acetyl group. The resulting diazoester dissociates into the corresponding diazonium salt. Trapping of the diazonium ion with 1-naphthol as an azo dye provides a useful means to identify the parent N-nitroso compound and to measure colorimetrically its rate of formation. The yields obtained in dilute aqueous nitrosation mixtures are lower than expected; the reasons for this finding are discussed. Preliminary results of animal experiments show that the N-nitroso compound is a directly acting carcinogen.

Acetanilides↗
PubMed1983

Mutagenic activity of a nitrosated early Maillard product: DNA synthesis (DNA repair) induced in HeLa S3 carcinoma cells by nitrosated 1-(N-L-tryptophan)-1-deoxy-D-fructose.

HeLa S3 cells in suspension were incubated at 37 degrees C with various concentrations of the Amadori compound 1-(N-L-tryptophan)-1-deoxy-D-fructose (Trp-Fru), of its nitrosated analogue NO-Trp-Fru and of sodium nitrite, for varying periods of time, and were assayed for viability (trypan blue exclusion test) and for intracellular DNA, RNA and protein synthesis. None of the compounds tested had any effect on cell viability, or on RNA and protein synthesis apart perhaps from a slightly inhibitory action. While Trp-Fru remained ineffective also as far as intracellular DNA synthesis was concerned, both NO-Trp-Fru and NaNO2 had a major effect on DNA synthesis. With NaNO2, stimulation of DNA synthesis occurred at concentrations above 1 mM in the growth medium, but with NO-Trp-Fru synthesis increased at concentrations below 1 microM. The excess DNA synthesis (i.e. synthesis above control activity) observed with NO-Trp-Fru and also with NaNO2 was due to DNA repair. This was verified by keeping the cells under conditions that prevented normal semi-conservative replication but permitted DNA repair ('unscheduled DNA synthesis'). Two major routes are suggested by which NO-Trp-Fru could damage DNA.

Cell Survival↗
PubMed1986

Identification of cholesterol as a mouse skin lipid that reacts with nitrogen dioxide to yield a nitrosating agent, and of cholesteryl nitrite as the nitrosating agent produced in a chemical system from cholesterol.

The skin lipids of mice exposed to nitrogen dioxide (NO2) and mouse skin lipids exposed in vitro to NO2 contain nitrosating agents (NSAs), that react with amines to produce nitrosamines. This situation represents a potential hazard of exposure to NO2. A principal NSA precursor in mouse skin lipids was purified by thin-layer and high-performance liquid chromatography. Each fraction was assayed by bubbling in NO2 and determining NSA. The precursor was identified as cholesterol on the basis of its chromatographic behavior and spectral properties. In a chemical system, cholesterol reacted with NO2 to give 13% yields of an NSA, which was identified from its spectral properties as the previously known compound, cholesteryl-3-beta-nitrite. These findings and the chromatographic behavior of a major NSA in the skin lipids of NO2-exposed mice suggested that this NSA was cholesteryl nitrite.

Animals↗
PubMed1996

S-nitrosation of serum albumin: spectrophotometric determination of its nitrosation by simple S-nitrosothiols.

The transfer of nitroso groups from S-nitroso-L-cysteine (1) and six other simple S-nitrosothiols to Cys 34 of bovine serum albumin (2) has been followed using Ellman's reagent, 5,5'-dithio-bis (2-nitrobenzoate) (3), to detect the resulting thiols. The described method utilizes the low reactivity of (3) with (2) and the high extinction coefficient of 2-nitro-5-thiobenzoate produced upon its reaction with thiols to follow the transfer of nitroso moieties at low concentrations where other procedures are not feasible. A second-order rate constant of 6400 M-1 s-1 obtained for the reaction of (2) with S-nitrosomercaptoethylamine is approximately 10 times faster than that for its reaction with (1), approximately 40 times faster than that for its reaction with S-nitrosoglutathione, and consistent with Cys 34 being located in a narrow crevice in close proximity to an anionic charge.

Animals↗
PubMed1994

Effects of aminosalicylates and immunosuppressive agents on nitric oxide-dependent N-nitrosation reactions.

Recent studies have demonstrated that nitric oxide (NO) rapidly and spontaneously decomposes in oxygenated solutions to generate potent N-nitrosating agents. These electrophilic substances have been shown to mediate mutagenesis and carcinogenesis via the formation of aliphatic and aromatic nitrosamines. We have also demonstrated that extravasated neutrophils and macrophages produce significant amounts of N-nitrosating agents derived exclusively from NO. During the course of these studies, we found that certain antioxidants, including 5-aminosalicylic acid (5-ASA), inhibited the leukocyte-mediated N-nitrosation reaction. Because 5-ASA and other anti-inflammatory and immunosuppressive drugs are used to treat inflammatory bowel disease, we wondered if any of these other compounds might also modulate N-nitrosation reactions in vitro. Therefore, the objectives of this study were to assess the ability of aminosalicylates and certain immunosuppressive agents to inhibit NO-dependent N-nitrosation of a model aromatic amine (2,3-diaminonaphthalene) and to determine whether this inhibitory activity correlated with their oxidation potential. We found that the concentrations necessary to inhibit the N-nitrosation reaction by 50% (IC50) were 25, 50 and 100 microM for 5-ASA, olsalazine (dimeric 5-ASA) and sulfasalazine, respectively. In contrast, sulfapyridine, 4-ASA, N-acetyl-5-ASA, 6-mercaptopurine, azathioprine, and methotrexate were either much less effective or inactive at inhibiting the N-nitrosation reaction. Although 5-ASA was able to fully scavenge the stable free radical 1,1-diphenyl-2-picrylhydrazyl, neither olsalazine nor sulfasalazine was found to be effective at scavenging this weak oxidant. We did find that olsalazine possessed an oxidation potential substantially less than that of sulfasalazine, suggesting that it may, in fact, scavenge more potent oxidizing agents such as the N-nitrosating agent. We conclude that 5-ASA and olsalazine inhibit NO-dependent N-nitrosation reactions by scavenging or decomposing the nitrosating agent(s). We propose that the secondary nitrogen unique to sulfasalazine interacts with the nitrosating agent to yield a secondary nitrosamine, thereby competing for N-nitrosation of our detector.

2-Naphthylamine↗
PubMed1989

The stability of the nitrosated products of indole, indole-3-acetonitrile, indole-3-carbinol and 4-chloroindole.

The nitrosation rates of indole-3-acetonitrile, indole-3-carbinol, indole and 4-chloroindole and the stability of their nitrosated products were investigated. Each of the nitrosated indole compounds was directly mutagenic to Salmonella typhimurium TA100 in the following order of potency: 4-chloroindole much greater than indole-3-carbinol greater than or equal to indole greater than indole-3-acetonitrile. Total N-nitroso determinations, carried out according to a modified method of Walters et al. (Analyst, Lond. 1978, 103, 1127), and Ames test results revealed that each of the indole compounds immediately formed mutagenic N-nitroso products upon nitrite treatment under acidic conditions. However, the nitrosation rates of indole and 4-chloroindole were higher than those of indole-3-acetonitrile and indole-3-carbinol. For indole-3-carbinol, indole-3-acetonitrile and indole, no change in the amount of nitrosated products was observed at increasing incubation times from about 15 up to 60 min. For 4-chloroindole the amount of nitrosated products decreased with increasing incubation times. In all cases the responses in the Ames test paralleled the amounts of nitrosated products. The stabilities of the nitrosated products of the indole compounds were investigated at pH 2 and 8. Both mutagenicity data and measurements by high-performance liquid chromatography using a photohydrolysis detector indicated that the nitrosation products of indole-3-acetonitrile, indole-3-carbinol and indole were more stable at pH 8 than at pH 2. Conversely, nitrosated 4-chloroindole was stable at pH 2 but not at pH 8. The pH 8 chromatograms showed a large nitrite peak. From this we hypothesized that the presence of free nitrite might be responsible for the stability of nitrosated indole-3-acetonitrile, indole-3-carbinol and indole at pH 8. Experiments confirmed the existence of an equilibrium between the nitrosated indole compound and the free indole compound plus nitrite.

Biotransformation↗
PubMed1998

Nitrosation by peroxynitrite: use of phenol as a probe.

Nitrosation is an important pathway in the metabolism of nitric oxide, producing S-nitrosothiols that may be critical signal transduction species. The reaction of peroxynitrite with aromatic compounds in the pH range of 5 to 8 has long been known to produce hydroxylated and nitrated products. However, we here present evidence that peroxynitrite also can promote the nitrosation of nucleophiles. We chose phenol as a substrate because the nitrosation reaction was first recognized during a study of the CO2-modulation of the patterns of hydroxylation and nitration of phenol by peroxynitrite (Lemercier et al., Arch. Biochem. Biophys. 345, 160-170, 1997). 4-Nitrosophenol, the principal nitrosation product, is detected at pH 7.0, along with 2- and 4-nitrophenols; 4-nitrosophenol becomes the dominant product at pH >/= 8.0. The yield of 4-nitrosophenol continues to increase even after pH 11.1, 1. 2 units above the pKa of phenol, suggesting that the phenolate ion, and not phenol, is involved in the reaction. Hydrogen peroxide is not formed as a by-product. The nitrosation reaction is zero-order in phenol and first-order in peroxynitrite, suggesting the phenolate ion reacts with an activated nitrosating species derived from peroxynitrite, and not with peroxynitrite itself. Under optimal conditions, the yields of 4-nitrosophenol are comparable to those of 2- and 4-nitrophenols, indicating that the nitrosation reaction is as significant as the nitration of phenolic compounds by peroxynitrite. Low concentrations of CO2 facilitate the nitrosation reaction, but excess CO2 dramatically reduces the yield of 4-nitrosophenol. The dual effects of CO2 can be rationalized if O=N-OO- reacts with the peroxynitrite anion-CO2 adduct (O=N-OOCO-2) or secondary intermediates derived from it, including the nitrocarbonate anion (O2N-OCO-2), the carbonate radical (CO*-3), and *NO2. The product resulting from these reactions can be envisioned as an activated intermediate X-N=O (where X is -OONO2, -NO2, or -CO-3) that could transfer a nitrosyl cation (NO+) to the phenolate ion. An alternative mechanism for the nitrosation of phenol involves the one-electron oxidation of the phenolate ion by CO*-3 to give the phenoxyl radical and the oxidation of O=N-OO- by CO*-3 to give a nitrosyldioxyl radical (O=N-OO*), which decomposes to give *NO and O2; the *NO then reacts with the phenoxyl radical giving nitrosophenol. Both mechanisms are consistent with the high yields of NO-2 and O2 during the alkaline decomposition of peroxynitrite and the potent inhibitory effect of N-3 on the nitrosation of phenol by peroxynitrite and peroxynitrite/CO2 adducts. The biological significance of the peroxynitrite-mediated nitrosations is discussed.

Carbon Dioxide↗
PubMedDate not supplied

Site-directed mutagenesis studies of human serum albumin define tryptophan at amino acid position 214 as the principal site for nitrosation.

The patterns of nitric oxide (NO) release from nitrosated bovine serum albumin (BSA), human serum albumin (HSA) and a number of recombinant HSA mutants were compared. All albumin species were nitrosated by incubation with acidified NO(2)(-). The pattern of NO release from BSA nitrosated with acidified NO(2)(-) was in agreement with previous reports which indicated that Cys-34 is the primary target for nitrosation in BSA. In contrast, the pattern of NO release from HSA nitrosated with acidified NO(2)(-) indicated that the primary nitrosation target was an amino acid residue other than Cys-34. Based on our initial findings and a previous report that tryptophan is a potential target for nitrosation by acidified NO(2)(-), several recombinant HSA mutants were synthesized in the yeast species Pichia pastoris. The following recombinant HSA species were produced: wild-type, C34S, W214L, W214E and W214L/Y411W HSA. Nitrosation of these mutants using acidified NO(2)(-) showed that Trp-214 is the primary nitrosation target in HSA. Mutation of Trp-214 led to an increase in Cys-34 nitrosation, indicating possible competition between these two residues for reaction with N(2)O(3), the reactive nitrosating species formed in aqueous acidified NO(2)(-) solutions.

Animals↗
PubMed1993

Mutagenic activity of peptides and the artificial sweetener aspartame after nitrosation.

Naturally occurring dipeptides, cholecystokinine (CCK, a tetrapeptide hormone) and the artificial sweetener aspartame were nitrosated for 10-30 min with 40 mM-nitrite (pH 3.5, 37 degrees C), and the resultant products examined for mutagenicity in Salmonella typhimurium TA100. Specific mutagenicities (net revertants per mumol precursor) spanned four orders of magnitude, with CCK being the most potent precursor (4700 revertants/mumol) followed by tryptophyl-tryptophan (Trp-Trp; 1000 revertants/mumol). Aspartame and glycyl-Trp (Gly-Trp) had intermediate activity (300 revertants/mumol), while Gly-Gly and methionyl-methionine were only weakly mutagenic (20 and 12 revertants/mumol, respectively). The dipeptides of aspartic acid, phenylalanine and tyrosine had no detectable mutagenicity (limits of detection 0.5, 40 and 5 revertants/mumol, respectively). Kinetic studies with aspartame and Gly-Trp suggested that the mutagenic products arose primarily from nitrosation of the primary amine rather than the amide or indole group. The mutagenicities of nitrosated aspartame and Gly-Trp were higher in TA100 than in TA98, and higher without than with enzymatic activation (S-9 mix) in both strains. The time-course study of Trp-Trp nitrosation showed the production of at least two mutagens: a potent but unstable mutagenicity was seen at very short nitrosation times and a more stable but weaker effect was obtained after more than 60 min of nitrosation. Not only the absolute specific mutagenicity but also the nitrite dependence of the nitrosation reaction and the stability of the nitroso product must be taken into account in determining the risk posed by endogenous nitrosation of foods in the human stomach. Under stomach conditions, nitrosation of the side-chains of certain Trp peptides would be expected to contribute more to the endogenous burden of nitrosated products than nitrosation of aspartame or Gly peptides.

Animals↗
PubMed2002

Reactivity of amino acids in nitrosation reactions and its relation to the alkylating potential of their products.

Nitrosation reactions of amino acids with an -NH(2) group [namely, six alpha-amino acids (glycine, alanine, alpha-aminobutyric acid, alpha-aminoisobutyric acid, valine, and norvaline); two beta-amino acids (beta-alanine and beta-aminobutyric acid), and one gamma-amino acid (gamma-aminobutyric acid)] were studied. Nitrosation was carried out in aqueous acid media, mimicking the conditions of the stomach lumen. The rate equation was r = k(3)(exp)[amino acid][nitrite](2), with a maximum k(3)(exp) value in the 2.3-2.7 pH range. The existence of an isokinetic relationship supports the argument that all the reactions share a common mechanism. A nitrosation mechanism is proposed, and the following conclusions are drawn: (i) Nitrosation reactions of amino acids with a primary amino group in acid media occur with dinitrogen trioxide as the main nitrosating agent. The finding that the nitrosation rate is proportional to the square of the nitrite concentration suggests that the yield of nitrosation products in the stomach would increase sharply with higher nitrate/nitrite intakes. (ii) Stomach hypochlorhydria could be a potential enhancer of in vivo amino acid nitrosation. (iii) The reactivity (k(3)()(exp)) [alpha-amino acids > beta-amino acids > gamma-amino acids] is the same as that found in a previous work for the alkylating potential of lactones formed from nitrosation products of the same amino acids. This implies that the nitrosation reactions of the most common natural amino acids are the most efficient precursors of the most powerful alkylating agents. (iv) The order of magnitude (10(7)-10(8) M(-1) s(-1)) of the bimolecular rate constants of nitrosation shows that such reactions occur through an encounter process.

Alkylating Agents↗
PubMed1988

Effects of nitrosation on the chemical composition and epidermal carcinogenicity of the nitrogen-rich fraction of a high-boiling coal liquid.

An 800-850 degrees F solvent-refined coal-II liquid was fractionated into chemical classes to obtain the aliphatic hydrocarbons, polycyclic aromatic hydrocarbons (PAH), nitrogen-containing polycyclic aromatic compounds (NPAC), and hydroxy-substituted PAH (hydroxy-PAH). The isolated NPAC fraction was refractionated by chemical class both before and after undergoing a nitrosation reaction. The nitrosated and non-nitrosated refractionated NPAC fractions were further subfractionated into secondary amine (pyrroles), primary amine-enriched (amino-PAH), and tertiary amine (azaarene) classes. The PAH and hydroxy-PAH composition of the NPAC fraction increased upon nitrosation, whereas the amino-PAH fraction composition decreased upon nitrosation. Nitrosation of standards indicated the amino-PAH compounds reacted to form parent PAH, chloro-substituted PAH, and methoxy-substituted PAH when analyzed by high-resolution gas chromatography (GC) and GC/mass spectrometry (MS). Some easily oxidized PAH compounds reacted to form ketones and quinones. All other standard reference compounds, chosen to be representative of the major chemical classes of compounds present in coal liquefaction materials, were unchanged by the nitrosation reaction. The amino-PAH of the nitrosated NPAC fraction reacted to form parent and some chloro-substituted PAH when analyzed by low-voltage direct-probe MS in addition to the methods given above. Epidermal carcinogenesis studies with the PAH, NPAC, nitrosated NPAC, and hydroxy-PAH fractions isolated from the 800-850 degrees F coal liquid indicated the PAH and NPAC were the most important determiners of skin carcinogenesis, with the PAH giving a higher response than the NPAC. The tumorigenicity of the NPAC was drastically reduced by nitrosation, probably due to the destruction of the amino-PAH upon nitrosation.

Animals↗