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Nitric oxide reacts rapidly with superoxide to form the strong nitrating agent peroxynitrite, which is responsible for much of the tissue damage associated with diverse pathophysiological conditions such as inflammation. The occurrence of free or protein-bound nitrotyrosine (NTYR) has been considered as evidence for in vivo formation of peroxynitrite. However, various agents can nitrate tyrosine, and their relative significance in vivo has not been determined due to lack of a sensitive method to analyze NTYR in tissue proteins and biological fluids. We have developed a new HPLC-electrochemical detection method to analyze NTYR in protein hydrolyzates or biological fluids. The sample is injected directly into a reversed-phase HPLC column and NTYR is subsequently reduced by a platinum column to 3-aminotyrosine, which is quantified with an electrochemical detector. The method is simple, selective, and sensitive (detection limit, 0.1 pmol per 20-microl injection). We have applied this method to compare in vitro the ability of various nitrating agents to form NTYR in bovine serum albumin and human plasma. Yields of NTYR formed in human plasma proteins incubated with 1 or 10 mM nitrating agent decreased in the following order: synthetic peroxynitrite > 3-morpholinosydonimine, a generator of both NO and superoxide > Angeli's salt, which forms nitroxyl anion (NO-) > spermine-NONOate, which releases NO > sodium nitrite plus hypochlorite, which forms the nitrating agent nitryl chloride (NO2Cl). A simple purification method using a C18 Sep-Pak cartridge is also described for analysis of free NTYR in human plasma.

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.

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.

International audience; Processed meat intake is associated with increased risk of colorectal cancer. This association may be explained by the endogenous formation of N-nitroso compounds (NOC). The hypothesis that meat intake can increase fecal NOC levels and colon carcinogenesis was tested in 175 Fischer 344 rats. Initiation was assessed by the number of aberrant crypt foci (ACF) in the colon of rats 45 days after the start of a high-fat bacon-based diet. Promotion was assessed by the multiplicity of ACF (crypts per ACF) in rats given experimental diets for 100 days starting 7 days after an azoxymethane injection. Three promotion studies were done, each in 5 groups of 10 rats, whose diets contained 7%, 14%, or 28% fat. Tested meats were bacon, pork, chicken, and beef. Fecal and dietary NOC were assayed by thermal energy analysis. Results show that feces from rats fed bacon-based diets contained 10-20 times more NOC than feces from control rats fed a casein-based diet (all p < 0.0001 in 4 studies). In bacon-fed rats, the amount of NOC input (diet) and output (feces) was similar. Rats fed a diet based on beef, pork, or chicken meat had less fecal NOC than controls (most p < 0.01). No ACF were detected in the colon of bacon-fed uninitiated rats. After azoxymethane injection, unprocessed but cooked meat-based diets did not change the number of ACF or the ACF multiplicity compared with control rats. In contrast, the bacon-based diet consistently reduced the number of large ACF per rat and the ACF multiplicity in the three promotion studies by 12%, 17%, and 20% (all p < 0.01). Results suggest that NOC from dietary bacon would not enhance colon carcinogenesis in rats.

Chronic inflammation induced by Helicobacter pylori infection has been associated with an increased risk of stomach cancer. We have analysed 167 stomach biopsies from 99 patients for H. pylori infection and immunohistochemically for the expression of inducible nitric oxide synthase (iNOS), catalase and superoxide dismutases (SODs) as markers of oxidative stress. Biopsies were graded as follows on the basis of histology: normal, superficial gastritis, variable severity of atrophic gastritis with or without intestinal metaplasia, and dysplasia. iNOS was detected in inflammatory cells in all types of gastritis with or without H. pylori infection and independently of its severity. In foveolar cells, iNOS was observed in approximately 25% of all biopsies showing any type of gastritis, but in a markedly higher proportion of dysplastic samples. Catalase and Mn-type SOD in inflammatory cells and catalase in foveolar cells were more frequently observed in marked atrophic gastritis biopsies than in less severe gastritis. Individual differences were found in the expression of these enzymes within groups with the same severity of gastritis. Prolonged oxidative stress in severe gastritis and dysplasia may play an important role in gastric carcinogenesis, through increased damage of DNA and tissue by reactive oxygen and nitrogen species.

Human urine is known to contain substances that strongly inhibit bacterial mutagenicity of aromatic and heterocyclic amines in vitro. The biological relevance of these anti-mutagens was examined by comparing levels of tobacco-related DNA adducts in exfoliated urothelial cells from smokers with the anti-mutagenic activity in corresponding 24-h urine samples. An inverse relationship was found between the inhibition of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-mutagenicity by urine extracts in vitro and two DNA adduct measurements: the level of the putatively identified N-(deoxyguanosine-8-yl)-4-aminobiphenyl adduct and the total level of all tobacco-smoke-related carcinogen adducts including those probably derived from PhIP. Urinary anti-mutagenicity in vitro appears thus to be a good indicator of the anti-genotoxicity exerted by substances excreted in urine, that protect the bladder mucosal cells (and possibly other cells) against DNA damage. These substances appear to be dietary phenolics and/or their metabolites because (i) the anti-mutagenic activity of urine extracts (n = 18) was linearly related to their content in phenolics; (ii) the concentration ranges of these substances in urine extracts were similar to those of various plant phenols (quercetin, isorhamnetin and naringenin) for which an inhibitory effect on the liver S9-mediated mutagenicity of PhIP was obtained; (iii) treatment of urines with beta-glucuronidase and arylsulfatase enhanced both anti-mutagenicity and the levels of phenolics in urinary extracts; (iv) urinary extracts inhibited noncompetitively the liver S9-mediated mutagenicity of PhIP as did quercetin, used as a model phenolics. Several structural features of the flavonoids were identified as necessary for the inhibition of PhIP and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxiline mutagenicity. Fractionation by reverse-phase HPLC and subsequent analysis of two urinary extracts, showed the presence of several anti-mutagenic substances and phenolics; more lipophilic phenolics displayed the highest specific inhibitory activity. This suggests that enzymatic conversion of dietary flavonoids into their more lipophilic and anti-mutagenic O-methylcatechol derivatives, as noted for quercetin, may occur in vivo in man. Onion, lettuce, apples and red wine are important sources of dietary flavonoids which are probably responsible for the anti-mutagenicity associated with foods and beverages. After HPLC fractionation of urinary extracts, the distribution profile of anti-mutagenic activity corresponded roughly to that of onion and wine extract combined. Our study strongly suggests that smokers ingesting dietary phenolics, probably flavonoids, are partially protected against the harmful effects by tobacco carcinogens within their bladder mucosal cells. This protective effect of dietary phenolics against the cancer of the bladder (and possibly other sites) should be verified and explored as a part of a chemoprevention strategy.

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.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.

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.

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 (range1-472 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 (pHor = 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 pH4 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.

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.

Various amounts of lyophilized beer were administered to rats dosed with proline and sodium nitrite. N-Nitrosoproline (NPRO) excreted in the 24-h urine was monitored as an index of endogenous nitrosation. In vitro formation of NPRO was determined after 15-min incubation of the same precursor solutions. Both in vivo and in vitro nitrosation of proline was inhibited in a dose-dependent fashion by lyophilized beers of different brands; the effects in vitro were most pronounced at pH below 4. The highest inhibitory effect was with beers with a high total polyphenolic content. Our results demonstrate that ingredients present in this widely consumed beverage inhibit endogenous nitrosation.

Extracts of betel nut (Areca catechu) were tested for their capacity to inhibit the endogenous formation of nitrosamines by measurement of the amount of urinary N-nitroso-L-proline (NPRO) following ingestion of sodium nitrate (300 mg) and L-proline (300 mg) by 2 volunteers. A water extract of the dried nuts, an ether extract containing mainly (+)-catechin and (-)-epicatechin, and a caffeine-precipitated n-butyl alcohol extract containing primarily proanthocyanidins (tannins) strongly reduced the endogenous formation of NPRO. An average of 14.7 and 10.9 micrograms NPRO (8 expts per individual) was excreted in the urine of the 2 volunteers over a 24-hour period following the intake of sodium nitrate and L-proline. The water extract and the proanthocyanidin (tannin)-containing extract, both of which contain the dose equivalent of one-quarter of a nut, reduced the excreted NPRO to background levels, which varied from 0.5 to 3.6 micrograms and from 0.6 to 2.1 micrograms (6 expts) in 24-hour urine samples from the 2 volunteers. These results may exemplify the way in which naturally occurring phenolics, which are ingested daily in relatively large quantities, could affect the endogenous formation of carcinogenic nitrosamines.

Measurements were made of the effects of phenolic compounds, some of which are present in the human diet, on the nitrosation of proline by nitrite to give N-nitrosoproline (NPRO). In vitro, resorcinol, catechin, p-nitrosophenol and phenol were catalysts and chlorogenic acid an inhibitor; guaiacol showed a marginal catalytic effect. Both the catalytic and the inhibiting effects were dependent on pH and on the concentration of phenolic compounds; catalysis by resorcinol and catechin was increased at optimal ratios of [nitrite]: [phenolic compound]. Endogenous nitrosation was examined in vivo by co-administration of nitrite, proline and a phenolic compound to rats and by monitoring the amount of NPRO excreted in the urine. Under similar experimental conditions, the catalytic effects observed in vivo decreased in the same order as those observed in vitro: resorcinol greater than p-nitroso-phenol greater than catechin greater than phenol greater than or equal to guaiacol; chlorogenic acid acted as an inhibitor. Catalysis and inhibition of N-nitrosation in rats in vivo appears to occur via mechanisms similar to those in vitro, although the effects in vivo were smaller. The implications of our findings for the endogenous formation of N-nitroso compounds and for variations in exposure due to different dietary constituents in humans are discussed.