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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↗
PubMed2003

The role of a formaldehyde dehydrogenase-glutathione pathway in protein S-nitrosation in mammalian cells.

Intracellular sulfhydryls, both protein and non-protein, are potential targets of nitric oxide-related species. S-Nitrosation of proteins can occur in vivo and can affect their activity. Metabolic pathways that regulate protein S-nitrosation are therefore likely to be biologically important. We now report that formaldehyde dehydrogenase, an enzyme that decomposes S-nitrosoglutathione, can indirectly regulate the level of cellular protein S-nitrosation. Nitrogen oxide donors induced high levels of protein S-nitrosation in HeLa cells and lower levels in Mutatect fibrosarcoma cells, as determined by Saville-Griess assay and Western-dot-blot analysis. Depletion of glutathione by treatment with buthionine sulfoximine markedly increased protein S-nitrosation in both cell lines. Glutathione depletion also increased cytokine-induced S-nitrosation in brain endothelial cells. Formaldehyde dehydrogenase activity was 2-fold higher in Mutatect than in HeLa cells. We downregulated formaldehyde dehydrogenase activity in Mutatect cells by stably expressing antisense RNA and short-interfering RNA. In these cells, both protein S-nitrosation and S-nitrosoglutathione levels were significantly enhanced after exposure to nitrogen oxide donors as compared to parental cells. Overall, a strong inverse correlation between total S-nitrosothiols and formaldehyde dehydrogenase activity was seen. Inhibition of glutathione reductase, the enzyme that converts oxidized to reduced glutathione, by dehydroepiandrosterone similarly increased protein S-nitrosation and S-nitrosoglutathione levels in both cell lines. Our results provide the first evidence that formaldehyde dehydrogenase-dependent decomposition of S-nitrosoglutathione plays a role in protecting against nitrogen oxide-mediated protein S-nitrosation. We propose that formaldehyde dehydrogenase and glutathione reductase participate in a glutathione-dependent metabolic cycle that decreases protein S-nitrosation following exposure of cells to nitric oxide.

Acetylcysteine↗
PubMed2003

HIF-1 alpha protein as a target for S-nitrosation.

Hypoxia-inducible factor-1 alpha (HIF-1 alpha) is a master regulator to sense decreased oxygen partial pressure. HIF-1 alpha stability regulation initiates a complex biological response that allows cells to act appropriately to meet patho-physiological situations of decreased oxygen availability. Recently, nitric oxide emerged as a messenger with the ability to stabilize HIF-1 alpha and to transactivate HIF-1 under normoxia. Considering that reactive nitrogen species are recognized for post-translation protein modifications, among others S-nitrosation, we asked whether HIF-1 alpha is a target for S-nitrosation. In vitro NO+ donating NO donors such as GSNO and SNAP provoked massive S-nitrosation of purified HIF-1 alpha. All 15 free thiol groups found in human HIF-1 alpha are subjected to S-nitrosation. Thiol modification is not shared by spermine-NONOate, a NO radical donating compound. However, spermine-NONOate in the presence of O(2)(-), generated by xanthine/xanthine oxidase, regained S-nitrosation, most likely via formation of a N(2)O(3)-like species. In vitro, S-nitrosation of HIF-1 alpha was attenuated by the addition of GSH or ascorbate. In RCC4 and HEK293 cells GSNO or SNAP reproduced S-nitrosation of HIF-1 alpha, however with a significantly reduced potency that amounted to modification of three to four thiols, only. Importantly, endogenous formation of NO in RCC4 cells via inducible NO synthase elicited S-nitrosation of HIF-1 alpha that was sensitive to inhibition of inducible NO synthase activity with N-monomethyl-L-arginine. NO-stabilized HIF-1 alpha was susceptible to the addition of N-acetyl-cysteine that destabilized HIF-1 alpha in close correlation to the disappearance of S-nitrosated HIF-1 alpha. In conclusion, HIF-1 alpha is a target for S-nitrosation by exogenously and endogenously produced NO.

Acetylcysteine↗
PubMed1995

Effects of superoxide on nitric oxide-dependent N-nitrosation reactions.

Recent studies have demonstrated that nitric oxide (NO) in the presence of superoxide (O2-) may mediate mutagenesis via the N-nitrosation of DNA bases followed by nitrosative deamination to yield their hydroxylated derivatives. We have found that phorbol myristate acetate (PMA)-activated extravasated rat neutrophils (PMNs) will N-nitrosate 2,3-diaminonaphthalene (DAN) to yield its highly fluorescent nitrosation product 2,3-naphthotriazole (triazole) via the L-arginine dependent formation of NO. Addition of SOD enhanced triazole formation suggesting that O2- production may inhibit the N-nitrosating activity and thus the mutagenic activity of inflammatory PMNs. The objective of this study was to assess the role of superoxide as a modulator of NO-dependent N-nitrosation reactions using PMA-activated PMNs as well as a chemically defined-system that generates both NO and superoxide. We found that PMA-activation of PMNs reduced found that PMA-activation of PMNs reduced the amount of N-nitrosation of DAN by approximately 64% when compared to non-stimulated cells (450 vs. 1250 nM). Addition of SOD but not inactivated SOD or catalase to PMA-activated PMNs enhanced the formation of triazole by approximately 4-fold (1950 nM). In addition, we found that the NO-releasing spermine/NO adduct (Sp/NO; 50 microM) which produces approximately 1.0 nmol NO/min generated approximately 8000 nM of triazole whereas the combination of Sp/NO and a superoxide generator (hypoxanthine/xanthine oxidase) that produces approximately 1.0 nmol O2-/min reduced triazole formation by 90% (790 nM). Addition of SOD but not catalase restored the N-nitrosating activity. We conclude that equimolar fluxes of superoxide react rapidly with NO to generate products that have only limited ability to N-nitrosate aromatic amino compounds and thus may have limited ability to promote mutagenesis via the nitrosative deamination of DNA bases.

2-Naphthylamine↗
PubMed1987

Magnetic semi-permeable polyethyleneimine microcapsules for monitoring of N-nitrosation in the gastrointestinal tract.

Semi-permeable polyethyleneimine (PEI) microcapsules were developed recently for trapping unstable electrophilic products in the gastrointestinal tract. Their N-nitrosation has been investigated in vitro and in vivo in the present study. At acid pH N-nitrosation was found to be linearly dependent on nitrite concentration, without a pH maximum. Up to 70% of the nitrosating agent was converted to N-nitroso products that were retained in the microcapsules and detected by a total N-nitroso assay procedure. Microcapsules prepared with different formulations (in order to vary membrane characteristics) provided a limit of detection in the range 1-10 nmol N-nitrosating agent and were also found to have a different capacity for N-nitrosation. Based on n.m.r. data it seems that such N-nitrosation is favoured by the incomplete protonation of this polyamine at acid pH and that nitrosation occurs at the least hindered secondary amine functions. Microcapsules were administered intragastrically in doses of 2 or 6 million to rats also receiving nitrite in the drinking water and were recovered magnetically from faeces. Relative to the maximum possible yield of N-nitrosated microcapsules that could have been excreted within the first 24 h, the excretion (maximum 4.9%) was found to depend on number of capsules administered and the time of administration relative to access to nitrite. Although performed with only a few animals, these preliminary data indicate a performance possibly superior to existing endogenous nitrosation indicators, and a dual purpose system to trap both nitrosating agents and their direct-acting DNA-damaging N-nitroso products.

Animals↗
PubMed1992

Levels of direct-acting mutagens, total N-nitroso compounds in nitrosated fermented fish products, consumed in a high-risk area for gastric cancer in southern China.

A high gastric cancer mortality in Fujian province (Peoples Republic of China) has been associated with the consumption of certain salted fermented fish products such as fish sauce (FS). We have investigated the levels and nature of N-nitroso compounds (NOC) and genotoxins present, before and after nitrosation, in 49 FS samples collected from villages in this high-risk area, pooled into six samples. The concentrations of total NOC before nitrosation ranged from 0.2 to 16 mumoles/l, and after nitrosation at pH 2 and pH 7, they rose by up to 4800- and 100-fold, respectively. In nitrosated samples, 40-50% of total NOC was not extractable into organic solvents; volatile N nitrosamines accounted for 1-2% and N-nitrosamino acids for 8-16% of total NOC. None of the FS samples exhibited genotoxic activity, but after nitrosation all were weakly active in the SOS chromotest. The highest SOS-inducing potency was observed with nitrosated ethyl acetate extracts of most samples. The formation of methylating agents was measured by incubation of nitrosated FS with DNA and subsequent analysis of 7-methylguanine adduct. 2 of the 6 nitrosated FS samples caused a slight increase in DNA methylation. 1 pooled home-made FS sample (the only one tested) contained tumour promoter-like substances, as measured by expression of certain EBV genes in Raji cells. HPLC fractionation of ethyl acetate extracts of FS samples allowed identification of three UV-absorbing peaks that, upon nitrosation, produced direct-acting genotoxins. This genotoxicity was partly ascribed to the formation of nitrite-derived arene diazonium cations that were characterized by a coupling reaction with N-ethyl-1-naphthylamine and thin-layer chromatography.

China↗
PubMed2005

Effect of added nucleophilic species on the rate of primary amino acid nitrosation.

The rate of primary amino acid nitrosation, both with and without the addition of nucleophilic species, has been studied using stopped-flow spectrophotometry. The rate of nitrosation in the presence of strong nucleophilic species such as thiocyanate and thiourea was shown to be much faster than nitrosation without the addition of a nucleophile. Rate constants were determined at 25 degrees C for reaction of the amino acids alanine, glycine, and valine with five common nitrosating agents. For the nitrosating agents nitrosyl chloride, nitrosyl bromide, and dinitrogen trioxide the rate of reaction was observed to approach the predicted encounter-controlled limit. However, for nitrosyl thiocyanate and S-nitrosothiourea nitrosation was found to be reaction-controlled. In the reaction-controlled regime, rate constants were found to increase with increasing electrophilic strength of the nitrosating agent, as measured by the parameter En, with a slope indicative of a product-like transition state. Activation energies were also measured, being around 10-30 kJ mol-1 for encounter-controlled rate constants, and 30-50 kJ mol-1 for reaction-controlled rate constants. Our results are discussed in the context of in vivo amino acid nitrosation, where it is proposed that the rate of nitrosation may be considerably greater than currently thought, due to the presence of nucleophilic species.

Alanine↗
PubMed2005

Nitric oxide-mediated nitrosation of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline potentiated by hemin and myeloperoxidase.

N-Nitrosamines formed by nitrosation of heterocyclic amines might initiate colon cancer in individuals consuming well-done red meat diets and with inflammatory conditions in their colon. This study investigates nitric oxide (NO)-mediated nitrosation of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and the influence of dietary (hemin) and inflammatory [NO, myeloperoxidase (MPO), and H(2)O(2)] components on nitrosation. Using the NO donor spermine NONOate (1.2 microM NO/min) at pH 7.4 with 0.005 mM MeIQx, a product due to NO autoxidation was at the limit of detection (1% of total radioactivity recovered by HPLC). Product formation increased 13- or 16-fold in the presence of 10 microM hemin or 85 nM MPO, respectively, with an in situ system for generating H(2)O(2) (glucose oxidase/glucose). The nitrosation product and its chloro derivative were analyzed by electrospray ionization mass spectrometry, and the product was determined to be 2-nitrosoamino-3,8-dimethylimidazo[4,5-f]quinoxaline (N-NO-MeIQx). Nitrosation by NO autoxidation was only detected at > or =1.2 microM NO/min and was not affected by H(2)O(2). Investigations with hemin determined minimum effective components necessary for potentiation: 1 microM hemin, 1 microM H(2)O(2)/min, and 0.012 microM NO/min. The reactive nitrogen oxygen species (RNOS) produced by hemin and MPO had a 4- and 3-fold, respectively, greater affinity for MeIQx than those produced by NO autoxidation. Test agents were used to characterize nitrosation. Results with catalase, SOD, azide, and NADH are consistent with multiple RNOS, the lack of peroxynitrite involvement in nitrosation, and peroxidatic potentiation by oxidative nitrosylation rather than nitrosation. Using phorbol ester stimulated human neutrophils, the formation of N-NO-MeIQx and its modification by test agents was consistent with MPO and not peroxynitrite. Thus, nitrosation of MeIQx and its potentiation by hemin and MPO provide a mechanism by which well-done red meat consumption and inflammation can generate N-nitroso compounds and initiate colon cancer under inflammatory conditions, such as colitis.

Animals↗
PubMed2001

Distinction between nitrosating mechanisms within human cells and aqueous solution.

The quintessential nitrosating species produced during NO autoxidation is N(2)O(3). Nitrosation of amine, thiol, and hydroxyl residues can modulate critical cell functions. The biological mechanisms that control reactivity of nitrogen oxide species formed during autoxidation of nano- to micromolar levels of NO were examined using the synthetic donor NaEt(2)NN(O)NO (DEA/NO), human tumor cells, and 4,5-diaminofluorescein (DAF). Both the disappearance of NO and formation of nitrosated product from DAF in aerobic aqueous buffer followed second order processes; however, consumption of NO and nitrosation within intact cells were exponential. An optimal ratio of DEA/NO and 2-phenyl-4,4,5,5-tetramethylimidazole-1-oxyl 3-oxide (PTIO) was used to form N(2)O(3) through the intermediacy of NO(2). This route was found to be most reflective of the nitrosative mechanism within intact cells and was distinct from the process that occurred during autoxidation of NO in aqueous media. Manipulation of the endogenous scavengers ascorbate and glutathione indicated that the location, affinity, and concentration of these substances were key determinants in dictating nitrosative susceptibility of molecular targets. Taken together, these findings suggest that the functional effects of nitrosation may be organized to occur within discrete domains or compartments. Nitrosative stress may develop when scavengers are depleted and this architecture becomes compromised. Although NO(2) was not a component of aqueous NO autoxidation, the results suggest that the intermediacy of this species may be a significant factor in the advent of either nitrosation or oxidation chemistry in biological systems.

Ascorbic Acid↗
PubMed1997

N-nitrosation of medicinal drugs catalysed by bacteria from human saliva and gastro-intestinal tract, including Helicobacter pylori.

Micro-organisms commonly present in human saliva and three DSM strains (Helicobacter pylori, Campylobacter jejuni and Neisseria cinerea), which can be isolated from the human gastro-intestinal tract, were assayed in vitro for their capacity to catalyse N-nitrosation of a series of medicinal drugs and other compounds. Following incubation at pH 7.2 in the presence of nitrate (or nitrite) for up to 24 (48) h, the yield of N-nitroso compounds (NOC) was quantified by HPLC equipped with a post-column derivatization device, allowing the sensitive detection of acid-labile and acid-stable NOC. Eleven out of the 23 test compounds underwent bacteria-catalysed nitrosation by salivary bacteria, the yield of the respective nitrosation products varying 800-fold. 4-(Methylamino)antipyrine exhibited the highest rate of nitrosation, followed by dichlofenac > metamizole > piperazine > five other drugs, whilst L-proline and L-thioproline had the lowest nitrosation rate. Ten drugs including aminophenazone, cimetidine and nicotine, did not inhibit bacterial growth, allowing transitory nitrite to be formed, but no N-nitroso derivatives were detected. Three drugs inhibited the proliferation of bacteria and neither nitrite nor any NOC were formed. Using metamizole as an easily nitrosatable precursor, two strains, Campylobacter jejuni and Helicobacter pylori, were shown to catalyse nitrosation in the presence of nitrite at pH 7.2. As compared to Neisseria cinerea used as a nitrosation-proficient control strain, H. pylori was 30-100 times less effective, whilst C. jejuni had intermediary activity. The results of our sensitive nitrosation assay further confirm that bacteria isolated from human sources, possessing nitrate reductase and/or nitrosating enzymes such as cytochrome cd1-nitrite reductase (Calmels et al., Carcinogenesis, 17, 533-536, 1996), can contribute to intragastric nitrosamine formation in the anacidic stomach when nitrosatable precursors from exogenous and endogenous sources are present.

Bacteria↗
PubMed1984

Catalysis and inhibition of N-nitrosation reactions.

A number of factors that can lead to an acceleration or inhibition of N-nitrosation reactions may play an important role in the formation of N-nitroso compounds in foods and other environmental samples and in the body. The anion thiocyanate is a particularly effective catalyst for nitrosamine formation. It occurs in normal human saliva, but at higher concentrations in the saliva of smokers. Although nitrosation reactions are accelerated in the normal manner with increasing temperature, rate enhancements are also observed in frozen systems, a phenomenon that may be important in frozen foods. Surfactants that form micellar aggregates can accelerate the nitrosation of hydrophobic amines. This may be important in foods in view of the presence of components such as lecithin, or in the body in view of the occurrence of bile acid micelles. Nitrosation reactions may also be accelerated in the presence of certain carbonyl compounds, thiols and nitrosophenols. A number of compounds readily convert nitrosating agents into innocuous products and hence inhibit nitrosation reactions. If reductones such as ascorbic acid are present, they may react much faster with nitrous acid than amines and therefore effectively scavenge the nitrosating agent. Rapid reaction of polyhydroxyphenols with nitrous acid is another example of how endogenous or exogenous nitrosation reactions may be inhibited. Many microorganisms can influence nitrosation reactions by converting nitrate to nitrite, a reaction that can take place in foods, but which is particularly important in the body.

Ascorbic Acid↗
PubMed1991

In-vitro testing and the carcinogenic potential of several nitrosated indole compounds.

4-chloro-methoxyindole is a naturally occurring compound in Vicia faba which can easily react with nitrite to form a N-nitroso compound. In this in vitro study, the potential genotoxic effects of nitrosated 4-chloro-6-methoxyindole and its structural analogue 4-chloroindole were evaluated for the first time by using both Salmonella and Chinese hamster V79 cells. Additionally, the inhibition of gap junctional intercellular communication in V79 cells by these compounds was determined; this is a validated parameter for tumor-promoting activity. Most assays were also performed with nitrosated indole-3-acetonitrile, a naturally occurring compound in brassicas. Both nitrosated chloroindoles were highly mutagenic to Salmonella typhimurium TA100 without the need of exogenous metabolic activation and were potent inducers of Sister Chromatid Exchanges. Nitrosated indole-3-acetonitrile generated the same effects, although at much higher concentrations. Equivocal results were obtained for the nitrosated chloroindoles in a forward mutation assay using the hypoxanthine guaninephosphoribosyltransferase locus. All nitrosated indole compounds significantly inhibited gap junctional intercellular communication. These results indicate that nitrosated chloroindoles and nitrosated indole-3-acetonitrile should be considered as mutagens and agents with potential tumor-promoting capacity.

Animals↗
PubMed2004

Amidine nitrosation.

The acidic nitrosation chemistry of nine acyclic secondary and tertiary amidines (Ph-N=C(R(1))NR(2)R(3); R(1) = H, CH(3), Ph; R(2), R(3) = H, Ph or (CH(3))(2) or C(CH(2))(4)) and several N-acylamidines was investigated. The principal nitrosation products were amides derived from the amino moiety and compounds derived from the benzenediazonium ion, which was independently trapped for quantitation in several cases. Tertiary amidines also produce nitrosamines in minor, but significant, yields. The benzamidines did not react, and the N-acylamidines hydrolyzed much more rapidly than they nitrosated. The data support the hypothesis that the reaction occurs by nitrosation on the imino nitrogen, followed by the addition of H(2)O to give a tetrahedral intermediate (alpha-hydroxynitrosamine) for which the main decomposition pathway generates an amide and a diazonium ion. In the case of the pyrrolidine-derived amidines, about 25% of the decomposition results in cleavage of the amine moiety, which nitrosates to give N-nitrosopyrrolidine. Pseudo-first-order rate constants for amidine nitrosation in aqueous acetic acid with excess nitrite at 25 degrees C ranged from (3 to 106) x 10(-5) s(-1), while the amidine basicity ranged over 5 pK(a) units. Rate constants corrected for amidine basicity showed the pyrrolidine derived amidines to be most reactive. The lack of benzamidine nitrosative reactivity is attributed to a very slow rate of H(2)O additon to the N-nitrosoamidinium ion and reversible nitrosation.

Amides↗
PubMed1988

Inhibitors of endogenous nitrosation. Mechanisms and implications in human cancer prevention.

Although the proof that N-nitroso compounds (NOC), a versatile class of carcinogens in animals, are also carcinogenic in man is lacking, humans are exposed through ingestion or inhalation to preformed NOC in the environment and through the endogenous nitrosation of amino precursors in the body. Activated macrophages can synthesize nitrate, nitrite and nitrosating agents that can form NOC. A number of bacterial strains isolated from human infections can produce NOC enzymatically from precursors at neutral pH. As a consequence endogenous nitrosation may occur at various sites of the body such as the oral cavity, stomach, urinary bladder, lungs, and at other sites of infection or inflammation. Since the demonstration by Mirvish et al. (1972) showing that ascorbate can reduce tumor formation in animals following feeding of nitrite plus amine, numerous substances to which humans are exposed have been identified and shown to inhibit formation of NOC in vitro, in animal models and in humans. Such inhibitors of nitrosation include vitamins C and E, phenolic compounds, and complex mixtures such as fruit and vegetable juices or other plant extracts. Nitrosation inhibitors normally destroy the nitrosating agents and thus act as competitors for the amino compound that serves as substrate for the nitrosating species. Independently, epidemiological studies have already established that fresh fruits and vegetables that are sources of vitamin C, other vitamins and polyphenols have a protective effect against cancers at various sites and in particular gastric cancer. Although the evidence that endogenously formed NOC are involved in human cancers is far from conclusive, it is suggestive and justifies preventive measures for reducing exposure to NOC. This article briefly reviews (i) the chemistry of NOC formation and inhibition, (ii) the studies in experimental animals which showed that inhibition of endogenous NOC synthesis leads to a reduction of toxic, mutagenic and carcinogenic effects, (iii) recent studies in humans where the degree of inhibition of endogenous NOC synthesis was directly quantified and lastly (iv) the contribution of nitrosation inhibitors to human cancer prevention.

Animals↗
PubMed2002

S-nitrosation of Ca(2+)-loaded and Ca(2+)-free recombinant calbindin D(28K) from human brain.

Calbindin D(28K) is noted for its abundance and specific distribution in mammalian brain and sensory neurons. It can bind three to five Ca(2+) ions and may act as a Ca(2+) buffer to maintain intracellular Ca(2+) homeostasis, but its exact role is still unknown. In the present study, mass spectrometric analysis reveals that the five cysteine residues in recombinant human brain calbindin D(28K) (rHCaBP) are derivatized with N-ethylmaleimide, consistent with the determination of 5.3 +/- 0.4 and 4.7 +/- 0.4 free thiols in the protein using the thiol-specific reagents 5,5'-dithiobis(2-nitrobenzoic acid) and 5-(octyldithio)-2-nitrobenzoic acid, respectively. The results of UV-vis and circular dichroism absorption, intrinsic fluorescence, and mass spectrometry measurements indicate that both Ca(2+)-loaded (holo) and Ca(2+)-free (apo) rHCaBP are S-nitrosated by S-nitrosocysteine (CysNO). The number of cysteine residues S-nitrosated in holorHCaBP and aporHCaBP are 2.6 +/- 0.05 and 3.4 +/- 0.09, respectively, as determined by the Saville assay. HolorHCaBP also undergoes S-nitrosation at one to three cysteine residues when exposed to S-nitrosoglutathione (GSNO), and Cys100 was found to be an S-nitrosation site by peptide mass mapping. Treatment of holorHCaBP with free NO resulted in a mass increase of 59 +/- 2 Da, corresponding to two NO adducts. Since up to four cysteine residues can be S-nitrosated in rHCaBP, it is proposed that the protein may act as a NO buffer or reservoir in the brain in a manner similar to serum albumin in blood. It is significant in this context that rHCaBP is found coexistent with nitric oxide synthase in cerebellum and that S-nitrosation varies with Ca(2+) binding, with S-nitrosation occurring to a greater extent in aporHCaBP than in the holoprotein. Furthermore, exposure of rHCaBP to either CysNO or GSNO also leads to rapid S-thiolation of Cys187. We demonstrate here for the first time that intrinsic protein fluorescence is a sensitive probe of protein S-nitrosation. This is due to efficient Förster energy transfer (R(0) approximately 17 A) between tryptophan donors and S-nitrosothiol acceptors.

Binding Sites↗
PubMed1995

S-nitrosation of serum albumin by dinitrosyl-iron complex.

The objective of this study was to identify a potential mechanism for S-nitrosation of proteins. Therefore, we assessed S-nitrosation of bovine serum albumin by dinitrosyl-iron-di-L-cysteine complex [(NO)2Fe(L-cysteine)2], a compound similar to naturally occurring iron-nitrosyls. Within 5-10 min, (NO)2Fe(L-cysteine)2 generated paramagnetic albumin-bound dinitrosyl-iron complex and S-nitrosoalbumin in a ratio of 4:1. Although S-nitroso-L-cysteine was concomitantly formed in low amounts, its concentration was not sufficient to account for formation of S-nitrosoalbumin via a trans-S-nitrosation reaction. Low oxygen tension did not affect S-nitrosation by the dinitrosyl-iron complex thus excluding the involvement of oxygenated NOx-species in the nitrosation reaction. Blockade of albumin histidine residues by pyrocarbonate, which prevented formation of dinitrosyl-iron-albumin complex, did not inhibit S-nitrosation of albumin. Thus, S-nitrosation of albumin by (NO)2Fe(L-cysteine)2 can proceed by direct attack of a nitrosyl moiety on the protein thiolate, without previous binding of the iron. We conclude that protein-bound dinitrosyl-iron complexes detected in high concentrations in certain tissues provide a reservoir of S-nitrosating species, e.g. low molecular dinitrosyl iron complexes.

Animals↗
PubMed1995

Nitric oxide-dependent N-nitrosating activity of rat pleural mesothelial cells.

Recent studies have demonstrated that nitric oxide (NO)-derived N-nitrosating agents may promote mutagenesis and carcinogenesis from the nitrosative deamination of DNA bases via the formation of nitrosamine intermediates. The objective of this study was to determine if pleural mesothelial cells (PMC) stimulated with proinflammatory cytokines could promote the N-nitrosation of a primary aromatic amine via the L-arginine-dependent formation of NO-derived N-nitrosation agents. N-nitrosating activity was determined by measuring the N-nitrosation of a model amine, 2,3-diaminonapthalene, to yield its fluorescent triazole (1-naptho-2,3-triazole) derivative. Results show that specific combinations of TNF, IL-1, interferon gamma, and LPS significantly increased N-nitrosating activity. There was a significant positive correlation between nitrite plus nitrate and triazole production. Triazole formation was inhibited by NG-nitro-L-arginine methyl ester, suggesting that triazole was derived from L-arginine-dependent formation of NO. These data indicate that PMC have the capacity to promote the N-nitrosation of primary aromatic amines via the formation of NO.

Animals↗
PubMed1997

Nitrosating agents: is peroxynitrite a likely candidate?

The reagents capable of effecting electrophilic and free radical nitrosation are briefly reviewed, together with the appropriate mechanisms, and in the light of these findings the possibilities of peroxynitrite/peroxynitrous acid acting as a nitrosating species are considered. The conclusion is that it is not to be expected that conventional direct nitrosation would occur. However, the very low yields of some nitrosation products can be accounted for in terms of nitrosation via the nitrogen oxide decomposition products of peroxynitrite. Thus NO could react with a radical substrate, or be converted to N2O3 (a well-known nitrosating species), whereas NO2 if formed can nitrosate via N2O4. A short post-script is added reviewing the Cu+-mediated reaction of S-nitrosothiols, generating (at low [thiol]) nitric oxide.

Copper↗