Analytical techniques for total N-nitroso compounds.
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Humans↗
Biomedical subjects
B Pignatelli
Publications and source records attributed to B Pignatelli.
Formation of 8-nitroguanine by the reaction of guanine with peroxynitrite in vitro.
Nitric oxide and superoxide anion, both formed in inflamed tissues, react rapidly to form the peroxynitrite anion (ONOO-), a strong oxidant which can initiate reactions characteristic of hydroxyl radical (HO.), nitronium ion (NO2+) and nitrogen dioxide radical (NO2.). Peroxynitrite, therefore, may cause DNA or tissue damage, contributing to the multistage carcinogenesis process. We have studied reactions of various bases, nucleosides or deoxynucleosides with peroxynitrite in vitro. Guanine reacted rapidly with peroxynitrite under physiological conditions and formed several substances, two of which were yellow, a characteristic of nitro and nitroso compounds. On the basis of chromatographic and spectral evidence we identified the major compound (which accounts for approximately 80% of all compounds formed) as 8-nitroguanine. Its formation was maximal at approximately pH 8 and increased dose-dependently with peroxynitrite concentration, but was not dependent on guanine concentration. The presence of ferric ions, which has been shown to catalyse nitration of tyrosine, did not affect nitration of guanine. 8-Nitroguanine could act as a specific marker for DNA damage induced by peroxynitrite in inflamed tissues.
Carcinogens↗
Effect of omeprazole on intragastric bacterial counts, nitrates, nitrites, and N-nitroso compounds.
Previous studies have suggested that profound inhibition of gastric acid secretion may increase exposure to potentially carcinogenic N-nitroso compounds. The aim of this study was to find out if the proton pump inhibitor omeprazole (20 mg daily) is associated with increased concentrations of potentially carcinogenic N-nitroso compounds in gastric juice. The volume of gastric contents, number of bacteria, and concentrations of nitrates, nitrites, and N-nitroso compounds was determined in gastric aspirates obtained after an overnight fast in 14 healthy volunteers (7M:7F) after one week of treatment with placebo, and one and two weeks' treatment with omeprazole. Median bacterial concentrations were 1.0 x 10(4) (range 5.0 x 10(3)-5.0 x 10(6)) colony forming units (CFU)/ml after one weeks' treatment with placebo and increased significantly to 4.0 x 10(5) (0-3.3 x 10(7)) CFU/ml after two weeks' treatment with omeprazole (p < 0.05). A similar increase was seen in the concentration of nitrate reducing bacteria. There was no difference in the volume of gastric aspirates after treatment with omeprazole when compared with placebo (65 (29-155) ml v 42 (19-194) ml). The concentration of N-nitroso compounds was 0.13 (0-1.0) mumol/l after two weeks of omeprazole, which was not significantly different from that seen with placebo (0.15 (0-0.61) mumol/l). There was also no increase in the concentrations of nitrates or nitrites. It is concluded that omeprazole (20 mg once daily) for two weeks in healthy volunteers is associated with gastric bacterial proliferation but does not increase concentrations of N-nitroso compounds.
Adult↗
Geographic association between urinary excretion of N-nitroso compounds and oesophageal cancer mortality in China.
Overnight urine samples were collected from approximately 60 male adults in each of 69 counties of China in 1989. Two specimens were collected from each subject--one after a loading dose of proline and ascorbic acid and another after a loading dose of proline only. Levels of N-nitrosamino acids and nitrate were measured in urine samples and correlated with cumulative mortality rates for subjects aged between 0 and 64 years in the 1970s. Oesophageal cancer mortality rates were positively and significantly associated with (i) urinary levels of excreted N-nitrosoproline (NPRO) (after proline and ascorbic acid loading or proline loading only), (ii) N-nitrososarcosine levels, and (iii) nitrosation potential (the decrease in the amount of urinary NPRO after adding ascorbic acid to the proline load). There were also positive correlations between the urinary level of NPRO or other N-nitrosamino acids and that of nitrate. The urinary excretion of nitrate was associated with consumption of various nitrate-rich vegetables. The results suggest that N-nitroso compounds (NOC) or other nitrite-derived carcinogens are implicated in the aetiology of oesophageal cancer in China.
Adult↗
Inhibition of nitrosation.
Humans are exposed through ingestion or inhalation to preformed N-nitroso compounds (NOC) in the environment and through the endogenous nitrosation of amino precursors in the body. Activated macrophages and bacterial strains isolated from human infections can enzymatically produce nitrosating agents and NOC 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, and at other sites of infection or inflammation. Numerous substances to which humans are exposed have been identified and shown to inhibit formation of NOC. Such inhibitors include vitamins C and E, certain 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. This article briefly reviews (a) the chemistry of NOC formation and inhibition; (b) the studies in experimental animals that showed that inhibition of endogenous NOC synthesis leads to a reduction of toxic, mutagenic, and carcinogenic effects; (c) recent studies in humans where the degree of inhibition of endogenous NOC synthesis was directly quantified; and (d) the possible contribution of nitrosation inhibitors to human cancer prevention.
Animals↗
Mutagens, N-nitroso compounds and their precursors in gastric juice from patients with and without precancerous lesions of the stomach.
This study examined whether elevated risk of gastric cancer is associated with high levels of total N-nitroso compounds (NOC), their precursors and nitrosation-dependent genotoxins in gastric juice (GJ). An improved method for quantifying total NOC was used and genotoxicity was assayed in E. coli. Results from patients (n = 210) with or without precancerous lesions of the stomach and living in three areas with up to 8-fold variations in gastric cancer risk (U.K., France, Colombia) were compared. The level of nitrite (range < 1-472 mumol/l) was found to increase with the pH of GJ from the three countries and was dependent on country of collection. The levels of NOC (range: < or = 0.01-8.0 mumol/l) in GJ were not affected by stomach histology and country of collection. NOC levels increased linearly with nitrite concentrations, but the slope of the regression line was greater for acidic GJ (pH < or = 4). These data together suggest that chemical nitrosation contributes at least as much as other nitrosation pathways to the intragastric formation of NOC. Acid-catalysed nitrosation of GJ in vitro increased the NOC concentration (range: 7-1332 mumol/l) up to several 1000-fold but this increase was not predictive of gastric cancer risk either by country or by stomach histology. After acid-catalysed nitrosation, direct genotoxicity (SOS-inducing potency) was significantly higher in GJ with original pH > 4 and highest in samples from Colombia. The results (a) provide no support that intragastric total NOC levels are elevated in subjects with precancerous stomach lesions or living in a high risk area for stomach cancer; (b) confirm that a high nitrite level and elevated pH in GJ are strongly associated, the level of nitrite being associated with precancerous stomach conditions only in Colombia; (c) reveal the presence of precursor compounds in GJ, that after nitrosation yield direct mutagens that probably contain NOC and other substances. As their concentrations were significantly higher in achlorhydric subjects and highest in Colombian patients, these data together provide support for a role of intragastrically formed nitrite-derived direct mutagens in gastric cancer aetiology.
Adult↗
High gastric juice ascorbic acid concentrations in members of a gastric cancer family.
Gastric juice ascorbic acid, total vitamin C, nitrite and N-nitroso-compound concentrations were determined in fasting gastric juice from four second generation members of a gastric cancer family, all of whom had Helicobacter pylori-associated chronic gastritis and intestinal metaplasia. Juice pH, nitrite and N-nitroso-compound concentrations were low. Juice ascorbate levels were comparable to those found in subjects with normal histology. The findings are contrary to our previous experience with juice ascorbate in H. pylori gastritis.
Adult↗
Intragastric mutagens and lowered anti-oxidant defence as risk factors for gastric cancer.
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Antioxidants↗
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↗
Endogenously formed N-nitroso compounds and nitrosating agents in human cancer etiology.
Humans are exposed to preformed N-nitroso compounds (NOC), but also to a wide range of precursors and nitrosating agents which can react in vivo to form potentially carcinogenic NOC and diazo compounds. Nitrite, nitrate and nitrosating agents can also be synthesized endogenously in enzymic reactions mediated by bacteria, activated macrophages and neutrophils. The latter two cell types generate, via the enzyme nitric oxide synthase, the nitric oxide radical that is involved in cytotoxicity, and is believed to be involved in formation of carcinogenic nitrosamines, DNA base deamination and oxidative damage. Thus endogenous NOC formation, DNA damage and gene mutations in humans could occur at various sites of the body such as the stomach and chronically infected or inflamed organs. Sensitive procedures to estimate the exposure of humans to NOC have been developed and applied in ecological and cross-sectional studies. These have shown that inhabitants of high-risk areas for stomach and esophageal cancer, patients with urinary tract infections (at risk for bladder cancer) and Thai subjects infected with liver fluke (at risk for cholangiocarcinoma) had significantly higher exposure to endogenous NOC. Clinical studies have examined the model of stomach carcinogenesis based on intragastric nitrosation, but the precise roles of bacterial overgrowth and of Helicobacter pylori infection in NOC synthesis and/or inducing oxidative stress in stomach mucosa remain to be clarified. Together these results support the role of NOC and other nitrite-derived mutagens in human cancer etiology, in particular when exposure starts early in life and persists over a long period.(ABSTRACT TRUNCATED AT 250 WORDS)
Adenoma, Bile Duct↗
Levels of nitrite, nitrate, N-nitroso compounds, ascorbic acid and total bile acids in gastric juice of patients with and without precancerous conditions of the stomach.
To determine the relevance of gastric juice factors to gastric carcinogenesis, 56 patients with unoperated stomachs undergoing endoscopy for dyspepsia had gastric juice aspirated and analysed for pH, ascorbic acid, total bile acids, nitrite, nitrate and total nitroso compounds (NOCs). Plasma was obtained for vitamin C estimation. Antral and body biopsies were assessed for gastritis, Helicobacter pylori, atrophy and intestinal metaplasia (IM). Patients with chronic atrophic gastritis (n = 17) had lower gastric juice ascorbic acid concentrations (P less than 0.001), higher pH (P less than 0.05) and higher incidence of H. pylori infection (P less than 0.001) than normal subjects (n = 12). Patients with reflux gastritis (n = 9) had higher total bile acids (P less than 0.01). Patients with chronic gastritis and IM (n = 11) had higher gastric juice pH (P less than 0.01) and total bile acid concentrations (P less than 0.05), and lower gastric ascorbic acid concentrations (P less than 0.01) than those with chronic gastritis and no IM (n = 24). In chronic gastritis, high nitrite concentrations were associated with high pH (P less than 0.01). However, there were no significant differences in plasma vitamin C or gastric nitrite, nitrate or total NOC concentrations in relation to gastric histology. We conclude that the premalignant condition IM is associated with H. pylori infection, low gastric ascorbic acid levels and elevated total bile acids, but not to elevation in nitrite or total NOCs in fasting gastric juice.
Adult↗
Studies in gastric carcinogenesis. V. The effects of ascorbic acid on N-nitroso compound formation in human gastric juice in vivo and in vitro.
The concentrations of nitrite, thermo- and acetic acid-labile TEA-responsive compounds (TACs) and N-nitroso compounds (NOCs) as a group were measured in human gastric juice collected just before and 1, 2 and 4 h after oral ingestion of 1 g ascorbic acid (AA) or 200 mg sodium nitrate, separately or in combination. Individual responses of gastric [nitrite] following ingestion of AA alone varied widely, with both decreases and increases being observed, and showed no correlation with gastric pH. While a mixed response was also noted for [NOC] and [TAC], substantial decreases were observed in 5/6 individuals with initial [NOC] greater than 0.2 microM and 3/3 individuals with initial [TAC] greater than 0.2 microM, implying that (i) AA effectively inhibited gastric nitrosation and (ii) a basal amount of NOCs and TACs was present in gastric juice which could not be lowered by AA ingestion. Statistical analysis indicated that global mean values of gastric [NOC] were significantly reduced (P less than 0.02) 1-4 h after ingestion of AA. Ingestion of 200 mg sodium nitrate alone resulted in increases in gastric [NOC], which in some cases were very substantial. While nitrosation appeared lower following ingestion of the same dose of nitrate in combination with 1 g AA, the difference from the effects of nitrate alone was not statistically significant. In aqueous buffer, pH 2.5, and in the presence of 1 mM AA, 50 microM nitrite was consumed with a t1/2 of 50 min only if molecular oxygen had first been removed from the system. In the presence of oxygen, no consumption of nitrite could be detected in 50 min, reflecting nitrite recycling (oxidation of nitric oxide to higher oxides of nitrogen and hydrolysis back to nitrite). It is likely that nitrite recycling occurring after collection of gastric juice accounted for the inconsistent responses of gastric nitrite following ingestion of AA. Incubation of human gastric juice, pH 2.5, in vitro in the presence of 50 microM sodium nitrite for 60 min resulted in an increase of [NOC] and [TAC] from 0.10 to 0.70 and 1.10 microM respectively. Nitrosation was efficiently inhibited by AA, 2.27 mM AA resulting in 87 and 100% inhibition respectively. Removal of oxygen from the reaction mixture did not have any significant effect on the extent of nitrosation in the presence or absence of AA.
Ascorbic Acid↗
Exposure to N-nitrosamines and other risk factors for gastric cancer in Costa Rican children.
The hypothesis that endogenous chemical nitrosation in the normal stomach in early life could play a crucial role in inducing chronic atrophic gastritis/intestinal metaplasia in later life was tested by applying the N-nitrosoproline (NPRO) test to 12-h urine samples from about 50 children (aged 8-14 years) living in high- and low-risk areas for stomach cancer. The median values of NPRO and the sum of four nitrosamino acids analysed were 0.28-0.84 micrograms/12 h and 0.75-1.75 micrograms/12 h, respectively. The NPRO level after proline intake was significantly higher in children from a high-risk area than in those from a low-risk area (p less than 0.04), and markedly reduced after ingestion of ascorbic acid and proline (p less than 0.05). Urinary nitrate level was lower than that of adults. NPRO levels on the day of proline intake, however, correlated well with nitrate levels (p less than 0.001), indicating that children in a high-risk area in Costa Rica have high endogenous nitrosation potential. Blood samples were also collected from about 300 children (aged 7-20 years) and analysed for antibodies against Campylobacter pylori, a suspected gastritis-causing bacteria. About 71% of children in both high- and low-risk areas for stomach cancer had antibodies. In addition, raw and cooked beans, which are consumed very frequently in Costa Rica, were collected from families in both areas and analysed for levels of nitrite/nitrate, total N-nitroso compounds and genotoxicity in the SOS chromotest.(ABSTRACT TRUNCATED AT 250 WORDS)
Adolescent↗
N-nitroso compounds, genotoxins and their precursors in gastric juice from humans with and without precancerous lesions of the stomach.
We are investigating the interrelationships between levels of total N-nitroso compounds (NOC), genotoxic activity (both before and after nitrosation), degree of bacterial colonization in gastric juice and degree of severity or absence of precancerous lesions of the stomach. The mean level of constitutive total NOC in gastric juice was similar in the different groups of patients, but it was higher in acidic gastric juice (n = 30) than in gastric juice at pH greater than 4.5 (n = 12). Acid-catalysed nitrosation of gastric juice in vitro increased the concentration of total NOC by up to several thousand fold, to a maximum of 1330 mumol/l. Genotoxicity, expressed as SOS-inducing potency per 100 microliters of gastric juice was measurable in only 20% of gastric juice samples tested. After acid-catalysed nitrosation, however, all samples showed genotoxic activity, the mean SOS-inducing potency being four to seven times greater than the corresponding constitutive value. There was no association between the mean SOS-inducing potency of gastric juice and the severity of precancerous lesions. The mean SOS-inducing potency of neutral or basic gastric juice was slightly greater than that of acidic samples. In a kinetic study on N-nitrosation of gastric juice in vitro, a mixture of amino and amido substrates was nitrosated; both qualitative and quantitative individual differences in nitrosatable substrates in gastric juice were seen. Fractionation of acidic, neutral and basic nitrosated gastric juice samples revealed a preponderance of nonvolatile, unknown NOC with varying polarities. The results of our study suggest that only pH determines the nature and level of precursors of NOC and of nitrosation-dependent genotoxins in gastric juice.
Adult↗
Exposure of humans to endogenous N-nitroso compounds: implications in cancer etiology.
Two sensitive procedures to quantitate human exposure to endogenous N-nitroso compounds (NOC) and/or methylating agents have been developed. One, the NPRO test, is based on the excretion of N-nitrosoproline (NPRO) and other N-nitrosoamino acids in the urine, that are measured as an index of endogenous nitrosation, following ingestion of precursors. The NPRO test has been applied to human subjects in clinical and epidemiological studies, and the kinetics and dietary modifiers of endogenous nitrosation have been investigated. Results obtained after application of the NPRO test to subjects at high risk for cancers of the stomach, esophagus, oral cavity and urinary bladder are summarized. In most instances, higher exposures to endogenous NOC were found in high-risk subjects, but individual exposure was greatly affected by dietary modifiers or disease state. Vitamin C efficiently lowered the body burden of intragastrically formed NOC. In experimental animals 3-methyladenine (3-MeAde) is excreted in urine following exposure to methylating NOC. Humans normally excrete 3-MeAde, the origin of which remains unknown. Recently developed analytical methodology permits large numbers of human urine samples to be analyzed and a wide variation is observed. Preliminary results suggest a weak correlation between basal NPRO excretion and background 3-MeAde excretion. Taken together, the results point to an etiological role of endogenously formed NOC in certain human cancers, and provide an interpretation of epidemiological findings that have shown protective effects of fruits and vegetables against several malignancies.
Adenine↗
Group-selective determination of total N-nitroso compounds in nitrate-containing human urine samples.
A group-selective method for the determination of total N-nitroso compounds (NOC) has been adapted for analysing human urine samples. Nitrate was first removed from urine by an anion-exchange procedure that prevented the significant loss of various added reference NOC and unidentified urinary NOC. The total NOC were then determined by injecting the urine sample (nitrate content less than 1 mmol l-1) or anion-exchange eluate into refluxing ethyl acetate containing either acetic acid for determining heat and acetic acid labile thermal energy analyser responsive compounds (TAC) or into hydrogen bromide for the determination of TAC and NOC. The nitrogen monoxide levels released were measured using thermal energy analysis with chemiluminescence detection, and the differnce between the two determinations represented the concentrations of NOC. The optimum conditions for preventing artefactual nitrosation in urine samples by the addition of sodium hydroxide or sulphamic acid without decomposition of NOC were determined. The influence of time and storage conditions on NOC stability was investigated. Fifteen urine samples collected from volunteers dosed with proline were analysed for total NOC and N-nitrosamino acids revealing a preponderance of unknown NOC. The determination of total NOC in human urine using this group-selective method offers a new approach to the estimation of human exposure to NOC and to isolate hitherto unknown NOC and their metabolites.
Chromatography, Gas↗
Human exposure to endogenous N-nitroso compounds: quantitative estimates in subjects at high risk for cancer of the oral cavity, oesophagus, stomach and urinary bladder.
A sensitive procedure to quantitate human exposure to endogenous N-nitroso compounds (NOC) has been developed. It is based on the excretion of N-nitrosoproline (NPRO) and other N-nitrosamino acids in the urine, which are measured as an index of endogenous nitrosation, following ingestion of precursors. The NPRO test has been applied to human subjects in clinical and epidemiological studies, and the kinetics and dietary modifiers of endogenous nitrosation have been investigated. Results obtained after application of the NPRO test to subjects at high risk for cancers of the stomach, oesophagus, oral cavity and urinary bladder are summarized. In most instances, higher exposures to endogenous NOC were found in high-risk subjects, but individual exposure was greatly affected by dietary modifiers or disease state. Vitamin C efficiently lowered the body burden of intragastrically formed NOC. The results point to an aetiological role of NOC in these human cancers and provide an interpretation of epidemiological findings that have shown protective effects of fruits and vegetables against several malignancies.
China↗
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↗