Mutagenicity of the reaction products of carbazole in the presence of nitrogen dioxide and nitrocarbazole

The mutagenicity of the photochemical reaction products of carbazole in the presence of nitrogen dioxide (NO2) and nitrocarbazole was investigated using a high-pressure mercury lamp (100 W). Samples extracted from the photochemical reaction products of carbazole with NO2 were more mutagenic than those of acridine and phenazine with NO2 for Salmonella typhimurium strain TA98 in the absence of S9 mix with a trend toward detoxification in the presence of the metabolic system. The mutagenicity of the photochemical reaction products of carbazole with NO2 were higher than those of the reaction products of carbazole with a mixture of NO2 and sulfur dioxide (SO2) and no irradiation. Mononitro- and dinitro-carbazole in the samples extracted from the reaction products were analyzed by mass spectrometry. It was suggested that mononitrocarbazole, which seemed to be weakly mutagenic, and dinitrocarbazole were readily formed by the reaction of carbazole with NO2, and that the other high-potency mutagens were formed by the photochemical reaction of carbazole with NO2 with irradiation by light.     

Effect of high-density extremely low frequency magnetic field on sister chromatid exchanges in mouse m5S cells

The induction of sister chromatid exchanges (SCEs) was evaluated in the cultured mouse m5S cells after exposure to extremely low frequency magnetic field (ELFMF; 5, 50 and 400 mT). Exposure to 5 mT and 50 mT ELFMF led to a very small increase in the frequency of SCEs, but no significant difference was observed between exposed and unexposed control cells. The cells exposed to 400 mT ELFMF exhibited a significant elevation of the SCE frequencies. There was no significant difference between data from treatments with mitomycin-C (MMC) alone and from combined treatments of MMC plus ELFMF (400 mT) at any MMC concentrations from 4 to 40 nM. These results suggest that exposure to highest-density ELFMF of 400 mT may induce DNA damage, resulting in an elevation of the SCE frequencies. We suppose that there may be a threshold for the elevation of the SCE frequencies, that is at least over the magnetic density of 50 mT.     

Effect of high-density extremely low frequency magnetic field on sister chromatid exchanges in mouse m5S cells

The induction of sister chromatid exchanges (SCEs) was evaluated in the cultured mouse m5S cells after exposure to extremely low frequency magnetic field (ELFMF; 5, 50 and 400 mT). Exposure to 5 mT and 50 mT ELFMF led to a very small increase in the frequency of SCEs, but no significant difference was observed between exposed and unexposed control cells. The cells exposed to 400 mT ELFMF exhibited a significant elevation of the SCE frequencies. There was no significant difference between data from treatments with mitomycin-C (MMC) alone and from combined treatments of MMC plus ELFMF (400 mT) at any MMC concentrations from 4 to 40 nM. These results suggest that exposure to highest-density ELFMF of 400 mT may induce DNA damage, resulting in an elevation of the SCE frequencies. We suppose that there may be a threshold for the elevation of the SCE frequencies, that is at least over the magnetic density of 50 mT.     

Effect of tumor promoter 12-O-tetradecanoyl-phorbol 13-acetate on induction of sister-chromatid exchanges in Chinese hamster V79 cells treated with mutagens

The effect of a tumor promoter, 12-O-tetradecanoyl-phorbol 13-acetate (TPA) alone and in combination with mitomycin C (MMC) or cyclophosphamide (CPP) on the induction of sister-chromatid exchanges (SCE) in Chinese hamster V79 cells was investigated. TPA alone at various doses and durations caused no increase of SCE frequency. MMC either at the dose of 0.03 or 0.003 μg/ml alone or in combination with TPA (2 μg/ml) all caused a significant increase of SCE frequencies. There was no difference in SCE frequencies between the cultures treated with MMC alone at 0.03 μg/ml and those treated with MMC plus TPA. However, cultures treated with MMC at 0.003 μg/ml plus TPA had significantly and consistently higher SCE frequencies than those treated with MMC alone at all durations. Treatment of CPP at 1 μg/ml activated by S9 mix caused significant increase of SCE frequencies. Surprisingly, the cultures treated with CPP, S9 mix plus TPA (2 μg/ml) caused a drastic reduction of SCE frequencies as compared to those treated with CPP and S9 mix only at all durations. These results indicate that TPA alone had no effect on SCE in V79 cells. TPA enhanced the SCE induction in V79 cells treated with MMC at a low dose, i.e. 0.003 μg/ml, but it inhibited SCE induction in cultures treated with the indirect mutagen CPP. Thus, TPA has no direct effect on genetic materials but it may indirectly alter the effects of a mutagen.     

Metabolite ratio of toluene-exposed rotogravure printing plant workers reflects individual mutagenic risk by sister chromatid exchanges

The study involved a group of 42 printing plant workers and a control group of 45 blood donors. At the working places, the ambient air-toluene concentration amounted from 141 to 328 mg/m(3). Sister chromatid exchanges (SCEs) were significantly elevated by three units in the exposed group. In this group, the concentration of urinary toluene metabolites was also considerably increased-hippuric acid was four times higher and the o-cresol and p-cresol fractions were twice as high. Results of toluene monitoring of ambient air- or blood-toluene concentrations did not show any relationships with individual SCE. While these SCE values revealed only a weak relationship with the corresponding hippuric acid data, a significant correlation with the cresols, which are known to be more genotoxic than hippuric acid, appeared in highly exposed workers. An attempt was made to consider the individual metabolic balance of toluene excretion products. For that reason individual cresol to hippuric acid ratios were calculated and related to corresponding SCE values. In all investigated subpopulations of the exposed group, this ratio correlated with SCE at a level of high significance. This strong interrelationship is a powerful argument for the genotoxic behavior of toluene. Furthermore, the individual metabolic balance, as a consequence of genetic polymorphism, should be considered in the discussion about genetic risk of toluene.     

Eicosanoid release from human bronchial epithelial cells upon exposure to toluene diisocyanate in vitro

Epithelial injury and inflammation are involved in airway hyperresponsiveness and asthma induced by toluene diisocyanate. In that isocyanates are insoluble and highly reactive compounds, bronchial epithelial cells may represent the most important target cells of their toxic effect. We hypothesized that damage to airway epithelium by toluene diisocyanate may result in the release of metabolites of arachidonic acid, which are known to promote inflammation and to alter epithelial cell function and airway smooth muscle responsiveness. To test this hypothesis we examined eicosanoid products in the culture media of bronchial epithelial cells exposed in vitro to 8 and 18 ppb toluene diisocyanate. Epithelial cells derived from human bronchi obtained at surgery were cultured to confluency on collagen-coated microporous membranes. Those cells, which expressed differentiated characteristics of epithelial cells (they showed keratin-containing filaments and had a cobblestone appearance), were alternatively exposed to toluene diisocyanate or air for 30 min in a specially designed in vitro chamber. The production of metabolites of arachidonic acid was assessed by measuring the release of immunoreactive products into the cell medium at the end of the exposure and during a 2 hr period after exposure. This method revealed a predominant isocyanate-induced release of immunoreactive 15-hydroxyeicosatetraenoic acid. Release rate of this compound tended to be dose-related and was associated with cell damage as assessed by the release of lactate dehydrogenase in the medium.     

Role of beta 2-adrenoceptors in the biological effect of autoimmune orchitis spleen cells

Autoimmune orchitis induced an increment in beta 2-adrenoceptor populations in intact mouse spleen lymphocytes. Normal and autoimmune lymphocytes incubated with soterenol increased the mechanical response of isolated atria. Autoimmune cells were more effective than normal cells in inducing this response. Soterenol or spleen cells alone did not modify the contractility at the concentration used. Inhibitors of beta 2-adrenoceptors of spleen cells completely blunted the reaction between soterenol and lymphocytes, while when atria were exposed to butoxamine, mechanical activity induced by soterenol plus lymphocytes was not affected. Cell-free supernatants of lymphocytes exposed to soterenol elicited the reaction in the same way as soterenol-treated lymphocytes. Direct contact of cells with the assay organ was not necessary. Inhibitors of cyclooxygenase on lymphocytes blocked the reaction of soterenol-treated lymphocytes on atria, while inhibitors of lipoxygenase(s) completely blocked the reaction of atria exposed to soterenol-stimulated lymphocytes or supernatants. These results suggest that soterenol reacts with beta 2-adrenoceptors of normal and autoimmune cells. From this reaction, soluble factors are released that in turn trigger stimulation of the atrial contractility as a consequence of the release of oxidative products of the lipoxygenase(s) pathway of arachidonic acid from atria. The high activity of atria in the presence of autoimmune spleen cells is probably related to the increment in number of beta 2-adrenoceptors of these cells.     

Interactions of nitric oxide and oxygen in cytotoxicity: Proliferation and antioxidant enzyme activities of endothelial cells in culture

Nitric oxide (NO) shows cytotoxicity, and its reaction products with reactive oxygen species, such as peroxynitrite, are potentially more toxic. To examine the role of O₂ in the NO toxicity, we have examined the proliferation of cultured human umbilical vein endothelial cells in the presence or absence of NO donor, ((Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)-amino]diazen-1-ium-1,2-diolate) (DETA-NONOate) (100–500 μM), under normoxia (air), hypoxia (< 0.04% O₂) or hyperoxia (88–94% O₂). It was found that the dose dependency on NONOate was little affected by the ambient O₂ concentration, showing no apparent synergism between the two treatments. We have also examined the effects of exogenous NO under normoxia and hyperoxia on the cellular activities of antioxidant enzymes involved in the H₂O₂ elimination, since many of them are known to be inhibited by NO or peroxynitrite in vitro. Under normoxia DETA-NONOate (500 μM) caused 25% decrease in catalase activity and 30% increases in glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities in 24 h. Under hyperoxia NO caused about 25% decreases in activities of catalase, glutathione reductase and glucose-6-phosphate dehydrogenase. The H₂O₂ removal rate by NO-treated cells was computed on the mathematical model for the enzyme system. It was concluded that the cellular antioxidant function is little affected by NO under normoxia but that it is partially impaired when the cells are exposed to NO under hyperoxia.     

Sister chromatid exchange in human chromosomes from normal individuals and patients with ataxia telangiectasia.

A new fluorescence plus Giemsa staining technique now makes the detection of sister-chromatid exchange (SCE) a relatively easy matter in cells containing 5-BrdU-substituted DNA. The technique has been applied to human cells to examine the distribution of SCE between different people and within different chromosomes. The results show: (1) That there were no large differences in the incidence of SCE between blood leukocyte chromosomes from male and female adults and newborn, and that similar frequencies were found in cells from two patients with ataxia telangiectasia which, nevertheless, showed the typical increases in chromosomal aberrations. (2) The distribution of SCE between chromosomes in the complement was found to be proportional to chromosome length, although the smaller chromosomes were under-represented, but not significantly so. (3) The distribution of SCE within chromosomes was nonrandom, with a deficiency in the centromeric and an excess in the mid-arm regions. There was no evidence for an excess of SCE in chromosome regions rich in AT DNA sequences. (4) The frequency of SCE is to some extent dependent of 5-BrdU concentration, but the influence of concentration is minimal within the range of from 1 to 160 muM. Human cells exposed over two cell cycles at these higher BrdU levels have around 14 SCE per cell-a frequency virtually identical with that observed in cultured cells from the Chinese hamster, wallaby, and rat kangaroo.     

Nitric oxide gas stimulates germination of dormant Arabidopsis seeds: use of a flow-through apparatus for delivery of nitric oxide

Nitric oxide (NO) is a gaseous free radical that reacts with O₂ in air and aqueous solution. NO donors have been widely used to circumvent the difficulties inherent in working with a reactive gas, but NO donors do not deliver NO at a constant rate for prolonged periods of time. Furthermore, some of the most commonly used NO donors produce additional, bioactive decomposition products. We designed and built an apparatus that allowed for the precise mixing of gaseous NO with air and the delivery of gas through sample vials at fixed rates. This experimental setup has the added advantage that continuous flow of gas over the sample reduces the buildup of volatile breakdown products. To show that this experimental setup was suitable for studies on the dormancy and germination of Arabidopsis thaliana seeds, we introduced vapors from water or sodium nitroprusside (SNP) into the gas stream. Seeds remained dormant when treated with water vapor, but gases generated by SNP increased germination to 90%. When pure NO was mixed with air and passed over dormant seeds, ∼ ∼30% of the seeds germinated. Because nitrite accumulates in aqueous solutions exposed to NO gas, we measured the accumulation of nitrite under our experimental conditions and found that it did not exceed 100 µM. Nitrite or nitrate at concentrations of up to 500 µM did not increase germination of C24 ecotype Arabidopsis seeds to more than 10%. These data support the hypothesis that NO participates in the loss of Arabidopsis seed dormancy, and they show that for some dormant seeds, exposure to exogenous NO is sufficient to trigger germination.     

Cytogenetic study in peripheral blood lymphocytes from asthmatic patients receiving continued therapy with theophylline.

Possible cytogenetic effects of theophylline have been investigated in asthmatic patients undergoing continuous therapy with this drug. Sister-chromatid exchanges (SCE), chromosome aberrations (CA) and proliferating rate indices (PRI) were evaluated in cultured peripheral blood lymphocytes from patients receiving theophylline alone, theophylline plus inhaled beta 2 adrenergic drugs or theophylline in combination with beta 2 adrenergic agents and corticoids. Two samples from each individual were obtained in order to perform a prospective study: before the theophylline medication (sample A) and at a time after the beginning of treatment (sample B). After treatment (66.3 +/- 37.8 days), an increase in SCE was observed without modifications either in PRI or in CA. Patients receiving beta 2 adrenergics or beta 2 adrenergics plus glucocorticoids before and during theophylline treatment, did not respond differently than those on theophylline alone.     

Atmospheric photochemical transformations enhance 1,3-butadiene-induced inflammatory responses in human epithelial cells: The role of ozone and other photochemical degradation products

Chemistry of hazardous air pollutants has been studied for many years, yet little is known about how these chemicals, once reacted within urban atmospheres, affect healthy and susceptible individuals. Once released into the atmosphere, 1,3-butadiene (BD) reacts with hydroxyl radicals and ozone (created by photochemical processes), to produce many identified and unidentified products. Once this transformation has occurred, the toxic potential of atmospheric pollutants such as BD in the ambient environment is currently unclear. During this study, environmental irradiation chambers (also called smog chambers), utilizing natural sunlight, were used to create photochemical transformations of BD. The smog chamber/in vitro exposure system was designed to investigate the toxicity of chemicals before and after photochemical reactions and to investigate interactions with the urban atmosphere using representative in vitro samples. In this study, we determined the relative toxicity and inflammatory gene expression induced by coupling smog chamber atmospheres with an in vitro system to expose human respiratory epithelial cells to BD, BDs photochemical degradation products, or the equivalent ozone generated within the photochemical mixture. Exposure to the photochemically generated products of BD (primarily acrolein, acetaldehyde, formaldehyde, furan and ozone) induced significant increases in cytotoxicity, IL-8, and IL-6 gene expression compared to a synthetic mixture of primary products that was created by injecting the correct concentrations of the detected products from the irradiation experiments. Interestingly, exposure to the equivalent levels of ozone generated during the photochemical transformation of BD did not induce the same level of inflammatory cytokine release for either exposure protocol, suggesting that the effects from ozone alone do not account for the entire response in the irradiation experiments. These results indicate that BDs full photochemical product generation and interactions, rather than ozone alone, must be carefully evaluated when investigating the possible adverse health effects to BD exposures. The research presented here takes into account that photochemical transformations of hazardous air pollutants (HAPs) does generate a dynamic exposure system and therefore provides a more realistic approach to estimate the toxicity of ambient air pollutants once they are released into the atmosphere.     

Tumor necrosis factor-alpha-dependent production of reactive nitrogen intermediates mediates IFN-gamma plus IL-2-induced murine macrophage tumoricidal activity.

We have previously established that IFN-gamma plus IL-2 induces murine macrophage tumoricidal activity. The purpose of this study was to identify the effector molecules that account for the IFN-gamma plus IL-2-induced macrophage cytotoxicity against P815 mastocytoma cells. ANA-1 macrophages and normal thioglycollate-elicited mouse peritoneal macrophages produced little or no detectable nitrite (NO2-) after incubation with IFN-gamma alone or IL-2 alone; however, IL-2 synergized with IFN-gamma for the production of NO2-. IFN-gamma plus IL-2 did not induce NO2- production or tumoricidal activity in ANA-1 macrophages that were cultured in medium devoid of L-arginine or in ANA-1 macrophages that were incubated with NG-monomethyl-L-arginine. As observed previously with ANA-1 macrophage tumoricidal activity, IL-4 inhibited IFN-gamma plus IL-2-induced, but not IFN-gamma plus LPS-induced, NO2- production. IL-4 also selectively decreased the ability of IFN-gamma and/or IL-2 to augment TNF-alpha mRNA expression in ANA-1 macrophages. Lastly, incubation of ANA-1 macrophages with anti-TNF mAb selectively inhibited the ability of IFN-gamma plus IL-2 to induce NO2- production and tumoricidal activity. These results indicate that IFN-gamma plus IL-2-induced tumoricidal activity is dependent upon the metabolism of L-arginine to reactive nitrogen intermediates, and they establish a role for TNF-alpha as a required intermediate for IL-2-dependent NO2- production and tumoricidal activity.     

Interactions of nitric oxide and oxygen in cytotoxicity: proliferation and antioxidant enzyme activities of endothelial cells in culture

Nitric oxide (NO) shows cytotoxicity, and its reaction products with reactive oxygen species, such as peroxynitrite, are potentially more toxic. To examine the role of O2 in the NO toxicity, we have examined the proliferation of cultured human umbilical vein endothelial cells in the presence or absence of NO donor, ((Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-++ +ium-1,2-diolate) (DETA-NONOate) (100-500 microM), under normoxia (air), hypoxia (< 0.04% O2) or hyperoxia (88-94% O2). It was found that the dose dependency on NONOate was little affected by the ambient O2 concentration, showing no apparent synergism between the two treatments. We have also examined the effects of exogenous NO under normoxia and hyperoxia on the cellular activities of antioxidant enzymes involved in the H2O2 elimination, since many of them are known to be inhibited by NO or peroxynitrite in vitro. Under normoxia DETA-NONOate (500 microM) caused 25% decrease in catalase activity and 30% increases in glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities in 24h. Under hyperoxia NO caused about 25% decreases in activities of catalase, glutathione reductase and glucose-6-phosphate dehydrogenase. The H2O2 removal rate by NO-treated cells was computed on the mathematical model for the enzyme system. It was concluded that the cellular antioxidant function is little affected by NO under normoxia but that it is partially impaired when the cells are exposed to NO under hyperoxia.     

Chemical induction of sister-chromatid exchanges in human lymphocytes treated in G0 prior to stimulation by different mitogens and revealed 72 h later in second division cells

Frequencies of sister-chromatid exchanges (SCE) were determined in second-division metaphases of human lymphocytes, exposed for 1 h during the G0 phase to mitomycin C (MMC) alone or to cyclophosphamide (CP) in the presence of S9 mix. The cells were then cultured for 72 h in the presence of phytohemagglutinin (PHA), concanavalin A (Con A), Wistaria floribunda (WFA) or Lens culinaris (LcH-A) extracts. Large differences in mitotic indices (MI) and cell-cycle kinetics were observed among cells subjected to the various treatments. However, in the controls as well as in the cultures submitted to a G0 mutagenic exposure, the yield of SCE was not influenced by the mitogenic agent and was, therefore, independent of the proliferation properties of the cultured lymphocyte population.     

Endogenous production of nitric oxide and effects of nitric oxide and superoxide on melanotrope functioning in the pituitary pars intermedia of Xenopus laevis.

Previous studies have focused on the immunohistochemical detection of a nitric oxide (NO)-cyclic 3',5'-monophosphate (cGMP) pathway in the brain and pituitary of the aquatic toad Xenopus laevis. We here investigate the endogenous production and possible involvement of NO signaling in the regulation of melanotrope cell activity in the pituitary pars intermedia of this amphibian. Using immunohistochemical staining of cultured cells with a polyclonal antiserum against inducible NO synthase (iNOS), immunoreactivity was observed both in melanotropes and in stellate-shaped cells. Part of these stellate-shaped cells is characterized as folliculo-stellate cells by their capacity of beta-Ala-Lys-N(epsilon)-AMCA uptake. Using chemiluminescence detection we demonstrate the presence of NO and reaction products like nitrite (NO(-)(2)) or peroxynitrite (ONOO(-)) in the incubation medium of cultured melanotropes. Bacterial lipopolysaccharide (LPS) stimulates the generation of NO and reaction products, the effect of which was blocked by S-methyl-l-thiocitrulline hydrochloride, a potent general NOS inhibitor. With [(3)H]lysine incorporation and a superfusion technique, it is shown that peptide release from melanotropes is stimulated by administration of superoxide dismutase (SOD), which was added to the superfusion medium to prevent scavenging of NO by superoxide anions. Pretreating the cells with the general NOS inhibitor l-nitroarginine methyl ester for 48 h attenuated the SOD-induced stimulation, but did not affect the stimulation by sodium nitroprusside (SNP) or 3-morpholinylsydnoneimine chloride (SIN-1), whereas hemoglobin blocked the combined effect of SOD plus NO donors. The soluble guanylate cyclase inhibitor 1H-[1,2, 4]oxadiazolo[4,3a]-quinoxaline-1-one did not inhibit but even significantly potentiated the effect of NO donors on peptide release without affecting the SOD-induced stimulation of peptide release. In addition to the previously described neuronal NOS (nNOS) immunoreactivity in nerve fibers in the pars intermedia of Xenopus, the present data reveal iNOS and nNOS as potential sources of endogenous NO production in cultured cells of the pars intermedia. Our study shows that also in nonmammalian vertebrates endogenous NO production may be physiologically relevant under conditions where protection against oxidative damage is needed. The endocrine cells of the pars intermedia themselves, as well as the folliculo-stellate cells, under such conditions may dispose of a protective mechanism against oxidative stress. The sensitivity of the endogenous NO production to LPS suggests that NO may also play a role during systemic inflammation.     

Anticlastogenic effects of black tea (World blend) and its two active polyphenols theaflavins and thearubigins in vivo in Swiss albino mice.

This study investigated the inhibition of cyclophosphamide (CP) and dimethylbenz(a)anthracene (DMBA) induced genetic damage by black tea (World blend) and its two active polyphenols theaflavins (TF) and thearubigins (TR) in Swiss albino mice as measured by chromosome aberrations (CA) and sister chromatid exchanges (SCE). Three different concentrations (5, 10 and 20%) of tea and a single dose of TF and TR were tested for their anticlastogenic effects against DMBA (50 mg/kg body weight) and CP (20 mg/kg for CA and 10 mg/kg for SCE). A significant decrease in CA was observed in all the three concentrations of tea extract plus DMBA treated groups when compared with the respective DMBA treated group alone. Similarly a significant decrease in CA was observed in all the three concentrations of tea extracts plus CP treated series when compared with the group treated with CP alone. In SCE assay, a significant decrease in SCE was observed in 5, 10 and 20% black tea extract plus CP and 10 and 20% tea extracts plus DMBA treated groups when compared with the CP or DMBA treated group alone. In the single dose of TF and TR treated groups a significant decrease in both CA and SCE was observed in both the TF and TR plus both the carcinogen treated groups when compared with their positive controls. The protective effects of black tea extracts were more significant than that of its two polyphenols. This study indicates that both black tea and its active polyphenols TF and TR have significant anticlastogenic effects in bone marrow cells of mice.     

Diagnostic insonation of extra utero human placentas: No effect of lymphocytic sister chromatid exchange

Summary Freshly delivered human placentas were exposed to ultrasound for 30 min using a diagnostic linear array unit. Blood was then drawn and cultured in the presence of bromodeoxyuridine, and the frequencies of sister chromatid exchanges (SCE) in the lymphocytes determined. There was no statistically significant difference in SCE frequencies between control and exposed cells; the frequencies of SCEs per cell ranged from 4.50 to 6.02 for control and from 4.66 to 6.10 for exposed cells in five separate experiments. Positive control mitomycin C treated cells were significantly affected, with more than 50 SCEs per cell.     

Cognac polyphenolic compounds increase bradykinin-induced nitric oxide production in endothelial cells

We recently reported that in vitro Cognac polyphenolic compounds (CPC) induce NO-dependent vasorelaxant effects and stimulate cardiac function. In the present study, we aim to investigate the effect of CPC on both nitric oxide (NO) and superoxide anions (O(2)(-)) production in cultured human endothelial cells. In addition, its effect on the bradykinin (BK)-induced NO production was also tested. The role and sources of O(2)(-) in the concomitant effect of BK plus CPC were pharmacologically determined. NO and O(2)(-) signals were measured using electron paramagnetic resonance technique using specific spin trappings. Both, CPC and BK induced an increase in NO production in human endothelial cells. The combination of both further enhanced NO release. The capacity of CPC plus BK to increase NO signal was blunted by the NO synthase inhibitor, N(G)-nitro-L-arginine methyl ester, and was enhanced in the presence either of superoxide dismutase or catalase. Moreover, CPC plus BK response was greater after inhibition of either NADPH oxidase by apocynin or xanthine oxidase by allopurinol but it was not affected by rotenone. CPC did not affect O(2)(-) level either alone or after its increase upon lipopolysaccharide treatment. Finally, the capacity of BK alone to increase NO was enhanced either by apocynin or allopurinol. Altogether, these data demonstrate that CPC is able to directly increase NO production without affecting O(2)(-) and enhances the BK-induced NO production in human endothelial cells. The data highlight the ability of BK to stimulate not only NADPH oxidase- but also xanthine oxidase-inhibitor sensitive mechanisms that reduce its efficiency in increasing NO either alone or in the presence of CPC. These results bring pharmacological evidence for vascular protection by CPC via its potentiating effect of BK response in terms of endothelial NO release.