To better understand the mechanism of in vivo toxicity of N-nitroso compounds (NNCs), the toxicity data of 80 NNCs related to their rat acute oral toxicity data (50% lethal dose concentration, LD50) were used to establish quantitative structure-activity relationship (QSAR) and classification models. Quantum chemistry methods calculated descriptors and Dragon descriptors were combined to describe the molecular information of all compounds. Genetic algorithm (GA) and multiple linear regression (MLR) analyses were combined to develop QSAR models. Fingerprints and machine learning methods were used to establish classification models. The quality and predictive performance of all established models were evaluated by internal and external validation techniques. The best GA-MLR-based QSAR model containing eight molecular descriptors was obtained with Q²loo = 0.7533, R² = 0.8071, Q²ext = 0.7041 and R²ext = 0.7195. The results derived from QSAR studies showed that the acute oral toxicity of NNCs mainly depends on three factors, namely, the polarizability, the ionization potential (IP) and the presence/absence and frequency of C⁻O bond. For classification studies, the best model was obtained using the MACCS keys fingerprint combined with artificial neural network (ANN) algorithm. The classification models suggested that several representative substructures, including nitrile, hetero N nonbasic, alkylchloride and amine-containing fragments are main contributors for the high toxicity of NNCs. Overall, the developed QSAR and classification models of the rat acute oral toxicity of NNCs showed satisfying predictive abilities. The results provide an insight into the understanding of the toxicity mechanism of NNCs in vivo, which might be used for a preliminary assessment of NNCs toxicity to mammals.

Nitrite-treated meat is a reported risk factor for colon cancer. Mice that ingested sodium nitrite (NaNO2) or hot dogs (a nitrite-treated product) showed increased fecal excretion of apparent N-nitroso compounds (ANC). Here, we investigated for the first time whether rats excrete increased amounts of ANC in their urine after they are fed NaNO2 and/or hot dogs. Rats were treated for 7 days with NaNO2 in drinking water or were fed hot dogs. Their 24 h urine samples were analyzed for ANC by thermal energy analysis on days 1-4 after nitrite or hot dog treatment was stopped. For two rats fed 480 mg NaNO2/L drinking water, mean urinary ANC excretion on days 1-4 was 30, 5.2, 2.5, and 0.8 nmol/day, respectively. For two to eight rats/dose given varied NaNO2 doses, mean urinary ANC output on day 1 increased from 0.9 (for no nitrite) to 37 (for 1000 mg NaNO2/L drinking water) nmol ANC/day. Urine samples of four rats fed 40-60% hot dogs contained 12-13 nmol ANC on day 1. Linear regression analysis showed highly significant correlations between urinary ANC excretion on day 1 after stopping treatment and varied (a) NaNO2 level in drinking water for rats fed semipurified or commercials diet and (b) hot dog levels in the diet. Some correlations remained significant up to 4 days after nitrite treatment was stopped. Urinary output of ANC precursors (compounds that yield ANC after mild nitrosation) for rats fed semipurified or commercial diet was 11-17 or 23-48 μmol/day, respectively. Nitrosothiols and iron nitrosyls were not detected in urinary ANC and ANCP. Excretion of urinary ANC was about 60% of fecal ANC excretion for 1 to 2 days after NaNO2 was fed. Administered NaNO2 was not excreted unchanged in rat urine. We conclude that urinary ANC excretion in humans could usefully be surveyed to indicate exposure to N-nitroso compounds.

It has been proposed that endogenously form N-nitroso compounds (NOCs) are partly responsible for the link between red meat consumption and colorectal cancer (CRC) risk. As nitrite has been indicated as critical factor in the formation of NOCs, the impact of replacing the additive sodium nitrite (E250) by botanical extracts in the PHYTOME project is evaluated.

A large number of drugs have been shown to react with nitrite to give genotoxic-carcinogenic N-nitroso compounds (NOC). However, the majority of drugs remain to be examined in this respect, among which calcium-channel blockers, all theoretically nitrosatable and widely used in the therapy of hypertension and other cardiovascular diseases. In this preliminary investigation, seven calcium-channel blockers have been examined either for their in vitro nitrosation according to the procedure recommended by the WHO, or for occurrence of liver DNA fragmentation, as detected by the Comet assay, in rats given by gavage 1/2 LD50 of the drug and 80 mg/kg of sodium nitrite. After 6h incubation the yields of NOC formed in vitro from nicardipine, nifedipine, nimodipine and nitrendipine ranged from 37 to 45% of the theoretical one, whereas the yields of NOC formed from diltiazem, gallopamil and verapamil ranged from 2 to 5%. In vivo, as compared with the effect of the same dose of the drug alone, a significant increase of both tail length and tail moment, indicative of an increased frequency of DNA single-strand breaks and alkali-labile sites, was produced in rat liver DNA by the administration with nitrite of gallopamil, nifedipine, nimodipine and nitrendipine, the ratio [tail length of drug+NaNO(2)/tail length of drug alone] being 3.2 for nimodipine, 3.1 for gallopamil 2.2 for nifedipine, and 2.1 for nitrendipine. Even if present, the increase in the degree of DNA fragmentation did not reach the statistical significance in rats given with nitrite nicardipine, diltiazem and verapamil. Further studies should be performed to investigate the formation of NOC in conditions simulating those occurring in the stomach of humans treated with a therapeutic dose, and to quantitate their genotoxic potency.

We previously showed that oxidative stress inhibits leukemia inhibitory factor (LIF) signaling by targeting JAK1, and the catalytic domains of JAK 1 and 2 have a cysteine-based redox switch. Thus, we postulated that the NO sibling and thiophylic compound, nitroxyl (HNO), would inhibit LIF-induced JAK-STAT3 activation. Pretreatment of human microvascular endothelial cells (HMEC-1) or neonatal rat cardiomyocytes with the HNO donors Angeli's salt or nitrosocyclohexyl acetate (NCA) inhibited LIF-induced STAT3 activation. NCA pretreatment also blocked the induction of downstream inflammatory genes (e.g. intercellular adhesion molecule 1, CCAAT/enhancer binding protein delta). The related 1-nitrosocyclohexyl pivalate (NCP; not a nitroxyl donor) was equally effective in inhibiting STAT3 activation, suggesting that these compounds act as thiolate targeting electrophiles. The JAK1 redox switch is likely not a target of acyloxy nitroso compounds, as NCA had no effect on JAK1 catalytic activity and only modestly affected JAK1-induced phosphorylation of the LIF receptor. However, pretreatment of recombinant human STAT3 with NCA or NCP reduced labeling of free sulfhydryl residues. We show that NCP in the presence of diamide enhanced STAT3 glutathionylation and dimerization in adult mouse cardiac myocytes and altered STAT3 under non-reducing conditions. Finally, we show that monomeric STAT3 levels are decreased in the Gαq model of heart failure in a redox-sensitive manner. Altogether, our evidence indicates that STAT3 has redox-sensitive cysteines that regulate its activation and are targeted by HNO donors and acyloxy nitroso compounds. These findings raise the possibility of new therapeutic strategies to target STAT3 signaling via a redox-dependent manner, particularly in the context of cardiac and non-cardiac diseases with prominent pro-inflammatory signaling.

Clonorchis sinensis, a high-risk pathogenic human liver fluke, provokes various hepatobiliary complications, including epithelial hyperplasia, inflammation, periductal fibrosis, and even cholangiocarcinogenesis via direct contact with worms and their excretory-secretory products (ESPs). These pathological changes are strongly associated with persistent increases in free radical accumulation, leading to oxidative stress-mediated lesions. The present study investigated C. sinensis infection- and/or carcinogen N-nitrosodimethylamine (NDMA)-associated fibrosis in cell culture and animal models. The treatment of human cholangiocytes (H69 cells) with ESPs or/and NDMA increased reactive oxidative species (ROS) generation via the activation of NADPH oxidase (NOX), resulting in augmented expression of fibrosis-related proteins. These increased expressions were markedly attenuated by preincubation with a NOX inhibitor (diphenyleneiodonium chloride) or an antioxidant (N-acetylcysteine), indicating the involvement of excessive NOX-dependent ROS formation in periductal fibrosis. The immunoreactive NOX subunits, p47phox and p67phox, were observed in the livers of mice infected with C. sinensis and both infection plus NDMA, concomitant with collagen deposition and immunoreactive fibronectin elevation. Staining intensities are proportional to lesion severity and infection duration or/and NDMA administration. Thus, excessive ROS formation via NOX overactivation is a detrimental factor for fibrogenesis during liver fluke infection and exposure to N-nitroso compounds.

The tumor suppressor gene breast cancer susceptibility gene 2 (BRCA2) is frequently mutated or epigenetically repressed in human cancer and has a significant role in the homologous recombination (HR) of DNA double-strand breaks (DSBs). Although N-nitrosodiethylamine (NDEA), N-nitrosodiethanolamine (NDELA) and N-nitrosodipropylamine (NDPA) have similar chemical structures and are able to induce DNA damage, they have varying carcinogenic risks. We hypothesized that the DNA damage repair pathways that are induced by these N-nitroso compounds (NOCs) may differ and that this may contribute to the genotoxic-carcinogenic effect of the NOCs. The present study aimed to characterize the formation of DSBs by NDEA, NDELA and NDPA and also to investigate whether BRCA2 is involved in the DNA damage response. The NOCs were observed to time-dependently induce DSBs and the expression of γ-H2AX in gastric cancer SGC7901 cells. It was observed that the DNA damage induced by NDEA, the most potent carcinogen, was not repaired as efficiently as that caused by NDELA or NDPA. The expression of BRCA2 and RAD51 was demonstrated to be inhibited by NDEA treatment but upregulated by NDELA or NDPA treatment. Furthermore, the knock down of BRCA2 expression impaired the DNA damage repair induced by NDELA or NDPA. The cells with this knock down exhibited an increased sensitivity to NDELA or NDPA treatment, but not to NDEA. These findings suggest that a BRCA2-mediated pathway contributes to differential DSB repair and sensitivity in response to NOC exposure and that it may be associated with the genotoxic-carcinogenic potential of NOCs.

Human serum and urine samples were analyzed for a suite of nitrosatable pesticides and potentially carcinogenic pesticide-associated N-nitroso (PANN) compounds. Formation of PANN compounds may occur in vivo after consumption of food or water containing trace amounts of nitrosatable pesticide residues and nitrate. Using a modified version of the Quick, Easy, Cheap, Effective, Rugged, and Safe (QuEChERS) method, nine nitrosatable pesticides and byproducts were extracted from serum and urine from 64 individuals from two different sample populations in Atlantic Canada: (i) Prince Edward Island, a region where nitrate and trace amounts of nitrosatable pesticides have been detected in groundwater; and (ii) Halifax, Nova Scotia, a non-agricultural urban area. Samples were then analyzed using ultra-high pressure liquid chromatography (UHPLC) coupled with high-resolution accurate mass (HRAM) single-stage orbitrap mass spectrometry (MS), which allows for semi-targeted analysis and tentative identification of a virtually limitless number of exposure biomarkers. Two nitrosatable target analytes, ethylenethiourea (ETU) and 3,5,6-trichloro-2-pyridinol (TCPy) were found in serum, while atrazine (ATR) and ETU were detected in urine. Five and six PANN compounds were tentatively identified in serum and urine, respectively. The two PANN compounds that were most frequently tentatively identified in serum were N-nitroso dimethoate (N-DIM) and N-nitroso omethoate (N-OME) with detection frequencies of 78% and 95%, respectively. This is the first biomonitoring study of its kind to investigate PANN compounds in human serum and urine.

Nitrate is converted to nitrite in the human body and subsequently can react with amines and amides in the gastrointestinal tract to form N-nitroso compounds (NOCs), which are known to be carcinogenic in animals. Humans can be exposed to nitrate via consumption of drinking water and diet, especially green leafy vegetables and cured meat. The contribution of nitrate from drinking water in combination with meat intake has not been investigated thoroughly. Therefore, in the present pilot study, we examined the effect of nitrate from drinking water, and its interaction with the consumption of white and processed red meat, on the endogenous formation of NOCs, taking into account the intake of vitamin C, a nitrosation inhibitor.

Consumption of nitrate-rich beetroot juice (BRJ) by athletes induces a number of beneficial physiological health effects, which are linked to the formation of nitric oxide (NO) from nitrate. However, following a secondary pathway, NO may also lead to the formation of N-nitroso compounds (NOCs), which are known to be carcinogenic in 39 animal species. The extent of the formation of NOCs is modulated by various other dietary factors, such as vitamin C. The present study investigates the endogenous formation of NOCs after BRJ intake and the impact of vitamin C on urinary NOC excretion. In a randomized, controlled trial, 29 healthy recreationally active volunteers ingested BRJ with or without additional vitamin C supplements for one week. A significant increase of urinary apparent total N-nitroso Compounds (ATNC) was found after one dose (5 to 47 nmol/mmol: p < 0.0001) and a further increase was found after seven consecutive doses of BRJ (104 nmol/mmol: p < 0.0001). Vitamin C supplementation inhibited ATNC increase after one dose (16 compared to 72 nmol/mmol, p < 0.01), but not after seven daily doses. This is the first study that shows that BRJ supplementation leads to an increase in formation of potentially carcinogenic NOCs. In order to protect athlete's health, it is therefore important to be cautious with chronic use of BRJ to enhance sports performances.

N-nitroso compounds represent a common type of environmental and endogenous DNA-damaging agents. After metabolic activation, many N-nitroso compounds are converted into a diazoacetate intermediate that can react with nucleobases to give carboxymethylated DNA adducts such as N3-carboxymethylthymidine (N3-CMdT) and O(4)-carboxymethylthymidine (O(4)-CMdT). In this study, we constructed non-replicative plasmids carrying a single N3-CMdT or O(4)-CMdT, site-specifically positioned in the transcribed strand, to investigate how these lesions compromise the flow of genetic information during transcription. Our results revealed that both N3-CMdT and O(4)-CMdT substantially inhibited DNA transcription mediated by T7 RNA polymerase or human RNA polymerase II in vitro and in human cells. In addition, we found that N3-CMdT and O(4)-CMdT were miscoding lesions and predominantly directed the misinsertion of uridine and guanosine, respectively. Our results also suggested that these carboxymethylated thymidine lesions may constitute efficient substrates for transcription-coupled nucleotide excision repair in human cells. These findings provided important new insights into the biological consequences of the carboxymethylated DNA lesions in living cells.

The recent outbreak of severe acute respiratory syndrome (SARS) warrants the search for effective antiviral agents to treat the disease. This study describes the assessment of the antiviral potential of nitric oxide (NO) against SARS coronavirus (SARS-CoV) strain Frankfurt-1 replicating in African Green Monkey (Vero E6) cells.

The beneficial cardiovascular effects of garlic have been reported in numerous studies. The major bioactive properties of garlic are related to organic sulfides. This study aimed to investigate whether garlic juice works exclusively due to its sulfur compounds or rather via the formation of new products of the nitroso-sulfide signaling pathway. Changes in isometric tension were measured on the precontracted aortic rings of adult normotensive Wistar rats. We evaluated NO-donor (S-nitrosoglutathione, GSNO)-induced vasorelaxation and compare it with effects of hydrogen sulfide (H2S)/GSNO and garlic/GSNO. Incubation with garlic juice increased the maximal GSNO-induced relaxation and markedly changed the character of the relaxant response. Although incubation with an H2S donor enhanced the maximal vasorelaxant response of GSNO, neither the absolute nor the relative relaxation changed over time. The mixture of GSNO with an H2S donor evoked a response similar to GSNO-induced relaxation after incubation with garlic juice. This relaxation of the H2S and GSNO mixture was soluble guanylyl cyclase (sGC) dependent, partially reduced by HNO scavenger and it was adenosine triphosphate-sensitive potassium channels (KATP) independent. In this study, we demonstrate for the first time the suggestion that H2S itself is probably not the crucial bioactive compound of garlic juice but rather potentiates the production of new signaling molecules during the GSNO-H2S interaction.

Endogenous formation of N-nitroso compounds (NOCs), which are known animal carcinogens, could contribute to human carcinogenesis but definitive evidence is still lacking. To investigate the relevance of NOCs in human colorectal cancer (CRC) development, we analyzed whole genome gene expression modifications in human colon biopsies in relation to fecal NOC exposure. We had a particular interest in patients suffering from intestinal inflammation as this may stimulate endogenous NOC formation, and consequently predispose to CRC risk. Inflammatory bowel disease (IBD) patients diagnosed with ulcerative colitis and irritable bowel syndrome patients without inflammation, serving as controls, were therefore recruited. Fecal NOC were demonstrated in the majority of subjects. By associating gene expression levels of all subjects to fecal NOC levels, we identified a NOC exposure-associated transcriptomic response that suggests that physiological NOC concentrations may potentially induce genotoxic responses and chromatin modifications in human colon tissue, both of which are linked to carcinogenicity. In a network analysis, chromatin modifications were linked to 11 significantly modulated histone genes, pointing towards a possible epigenetic mechanism that may be relevant in comprehending NOC-induced carcinogenesis. In addition, pro-inflammatory transcriptomic modifications were identified in visually non-inflamed regions of the IBD colon. However, fecal NOC levels were slightly but not significantly increased in IBD patients, suggesting that inflammation did not strongly stimulate NOC formation. We conclude that NOC exposure is associated with gene expression modifications in the human colon that may suggest a potential role of these compounds in CRC development.

Nitro-substituted 1,3-benzothiazinones (nitro-BTZs) are mechanism-based covalent inhibitors of Mycobacterium tuberculosis decaprenylphosphoryl-β-D-ribose-2'-oxidase (DprE1) with strong antimycobacterial properties. We prepared a number of oxidized and reduced forms of nitro-BTZs to probe the mechanism of inactivation of the enzyme and to identify opportunities for further chemistry. The kinetics of inactivation of DprE1 was examined using an enzymatic assay that monitored reaction progress up to 100 min, permitting compound ranking according to kinact/Ki values. The side-chain at the 2-position and heteroatom identity at the 1-position of the BTZs were found to be important for inhibitory activity. We obtained crystal structures with several compounds covalently bound. The data suggest that steps upstream from the covalent end-points are likely the key determinants of potency and reactivity. The results of protein mass spectrometry using a 7-chloro-nitro-BTZ suggest that nucleophilic reactions at the 7-position do not operate and support a previously proposed mechanism in which BTZ activation by a reduced flavin intermediate is required. Unexpectedly, a hydroxylamino-BTZ showed time-dependent inhibition and mass spectrometry corroborated that this hydroxylamino-BTZ is a mechanism-based suicide inhibitor of DprE1. With this BTZ derivative, we propose a new covalent mechanism of inhibition of DprE1 that takes advantage of the oxidation cycle of the enzyme.

We studied the effects of diethylamine/NO complex (DEA/NO) and S-nitroso-N-acetylpenicillamine (SNAP), relative to those of sodium nitroprusside (SNP) and nitroglycerin (NTG), on mean arterial pressure (MAP), mean circulatory filling pressure (MCFP), arterial resistance (Ra), venous resistance (Rv), heart rate (HR), cardiac output (CO) and stroke volume (SV) in groups of Inactin-anaesthetized rats pre-treated with i.v. mecamylamine (3.7 micromol kg(-1)) and noradrenaline (6.8 nmol kg(-1) min(-1)). Doses of each that reduced MAP by 30%, 80% and the lowest dose that maximally reduced MAP were examined to allow a comparison of the compounds' dilator actions at equivalent effective depressor doses. DEA/NO (4, 32 and 256 microg kg(-1) min(-1)), SNAP (4, 32 and 256 microg kg(-1) min(-1)) and SNP (8, 32 and 128 microg kg(-1) min(-1)) caused similar dose-dependent reductions in MAP and Ra, and increases in CO and SV. NTG (0.2, 0.8 and 6.4 microg kg(-1) min(-1)) dose-dependently reduced Ra, and increased CO and SV, but lowered MAP only at the highest dose. DEA/NO, SNAP and SNP but not NTG lowered MCFP with efficacy: DEA/NO > SNAP > SNP. All four drugs reduced Rv with efficacy: DEA/NO approximately equal to SNAP > SNP approximately equal to NTG. Therefore, all compounds lowered Ra and Rv. DEA/NO, SNAP and SNP but not NTG reduced MCFP. The pharmacological profiles of DEA/NO and SNAP resemble SNP more than NTG.

Cell aging depends on the rate of cumulative oxidative and nitrosative damage to DNA and proteins. Accumulated data indicate the involvement of protein S-nitrosylation (SNO), the nitric oxide (NO)-mediated posttranslational modification (PTM) of cysteine thiols, in different brain disorders. However, the changes and involvement of SNO in aging including the development of the organism from juvenile to adult state is still unknown. In this study, using the state-of-the-art mass spectrometry technology to identify S-nitrosylated proteins combined with large-scale computational biology, we tested the S-nitroso-proteome in juvenile and adult mice in both cortical and striatal regions. We found reprogramming of the S-nitroso-proteome in adult mice of both cortex and striatum regions. Significant biological processes and protein-protein clusters associated with synaptic and neuronal terms were enriched in adult mice. Extensive quantitative analysis revealed a large set of potentially pathological proteins that were significantly upregulated in adult mice. Our approach, combined with large scale computational biology allowed us to perform a system-level characterization and identification of the key proteins and biological processes that can serve as drug targets for aging and brain disorders in future studies.

Previous studies show that linking acetylated glucosamine to S-nitroso-N-acetyl-D,L-penicillamine (SNAP) stabilizes the molecule and causes it to elicit unusually prolonged vasodilator effects in endothelium-denuded, isolated rat femoral arteries. Here we studied the propanoyl (SNPP; 3 carbon side-chain), valeryl (SNVP; 5C) and heptanoyl (SNHP; 7C) N-substituted analogues of SNAP (2C), to further investigate other molecular characteristics that might influence chemical stability and duration of vascular action of S-nitrosothiols. Spectrophotometric analysis revealed that SNVP was the most stable analogue in solution. Decomposition of all four compounds was accelerated by Cu(II) and cysteine, and neocuproine, a specific Cu(I) chelator, slowed decomposition of SNHP. Generation of NO from the compounds was confirmed by electrochemical detection at 37 degrees C. Bolus injections of SNAP (10 microl; 10(-8)-10(-3) M) into the perfusate of precontracted, isolated rat femoral arteries taken from adult male Wistar rats (400-500 g), caused concentration-dependent, transient vasodilatations irrespective of endothelial integrity. Equivalent vasodilatations induced by SNVP and SNHP were transient in endothelium-intact vessels but failed to recover to pre-injection pressures at moderate and high concentrations (10(-6)-10(-3) M) in those denuded of endothelium. This sustained effect (> 1 h) was most prevalent with SNHP and was largely reversed by the NO scavenger, haemoglobin. We suggest that increased lipophilicity of SNAP analogues with longer sidechains facilitates their retention by endothelium-denuded vessels; subsequent slow decomposition within the tissue generates sufficient NO to cause prolonged vasodilatation. This is a potentially useful characteristic for targeting NO delivery to areas of endothelial damage.

We evaluated the anti-mycobacterial effect of a flavonoid 5,7-dihydroxy-2-(4-hydroxyphenyl) 4H-chromen-4-one (1) and two pyrimidines, 4-hydroxy-2-dimethylamino-5-nitroso-6-aminopyrimidine (2) and 2-chloro-5-n-nonylpyrimidine (3) in vitro against Mycobacterium tuberculosis (M. tuberculosis, H37Ra) and Mycobacterium avium (M. avium), using a Microplate Alamar Blue Assay (MABA). The effects of the compounds 1-3 in combination with first- and second-line anti-TB drugs isoniazid, rifampicin, cycloserine, and clarithromycin on the growth of M. tuberculosis and M. avium were also evaluated in in vitro assays. As a single agent, compounds 1 and 2 exhibited modest activity while compound 3 was the most effective against M. tuberculosis and M. avium. When compounds 1-3 were evaluated at lower than 50% of their inhibitory concentrations in a two-drug combination with isoniazid or rifampicin, they showed additive to synergistic interactions. This inhibitory effect was improved when each of the three compounds was tested together in a three-drug combination with two of the first-line anti-TB drugs. Compounds 1-3 also demonstrated strong synergistic interaction in combination with cycloserine and clarithromycin in inhibiting the growth of M. tuberculosis and M. avium, respectively. This study demonstrated that compounds 1-3 have potential to be developed as effective anti-TB agents with combined use.

1. The effects of two chemically unrelated nitric oxide (NO)-releasing compounds were studied on prostacyclin production in lipopolysaccharide (LPS)-stimulated human umbilical vein endothelial cells (HUVECs). The cells expressed cyclooxygenase-2 (COX-2) protein and produced prostacyclin by NS-398-sensitive manner suggesting that prostacyclin production derives principally by COX-2 pathway. 2. A novel NO-releasing oxatriazole derivative GEA 3175 (1-30 microm) inhibited LPS-induced production of prostacyclin in HUVECs in a dose-dependent manner being more potent than the earlier known NO-donor S-nitroso-N-acetylpenicillamine (SNAP). 3. The effects of the two NO-donors on prostacyclin synthesis were reversed when red blood cells were added into the culture indicating that the effects are due to NO released from the compounds. 4. Addition of exogenous arachidonic acid into the culture did not alter the inhibitory action of NO-donors suggesting that phospholipases are not the target of action of NO. 5. The NO-donors did not inhibit prostacyclin production in the presence of a selective COX-2 inhibitor NS-398. These data suggest that NO affects COX-2 pathway rather than has an overall effect on cyclooxygenases. 6. NO-releasing compounds did not alter the level of COX-2 protein expression in LPS-treated HUVECs as measured by Western blot analysis. 7. The results suggest that NO-donors inhibit the activity of COX-2 in human endothelial cells. A link between NO and the regulation of eicosanoid synthesis could represent an important mechanism in controlling vascular and inflammatory responses in pathophysiological states and during treatment with nitrovasodilators.