Concentrations Of N-nitroso Compounds Research Articles

Dietary Nitrosamines from Processed Meat Intake as Drivers of the Fecal Excretion of Nitrosocompounds.

Diet is one of the main exogenous sources of potentially carcinogenic nitrosamines (NAs) along with tobacco and cosmetics. Several factors can affect endogenous N-nitroso compounds (NOCs) formation and therefore the potential damage of the intestinal mucosa at initial colorectal cancer stages. To address this issue, 49 volunteers were recruited and classified according to histopathological analyses. Lifestyle and dietary information were registered after colonoscopy. The mutagenicity of fecal supernatants was assayed by a modified Ames test. Fecal heme-derived NOCs and total NOC concentrations were determined by selective denitrosation and chemiluminescence-based detection. Results revealed processed meats as the main source of dietary nitrites and NAs, identifying some of them as predictors of the fecal concentration of heme-derived and total NOCs. Furthermore, increased fecal NOC concentrations were found as the severity of colonic mucosal damage increased from the control to the adenocarcinoma group, these concentrations being strongly correlated with the intake of the NAs N-nitrosodimethylamine, N-nitrosopiperidine, and N-nitrosopyrrolidine. Higher fecal NOC concentrations were also noted in higher fecal mutagenicity samples. These results could contribute to a better understanding of the importance of modulating dietary derived xenobiotics as related with their impact on the intestinal environment and colonic mucosa damage.

Daily berry consumption attenuates β-catenin signalling and genotoxicity in colon carcinoma cells exposed to faecal water from healthy volunteers in a clinical trial

Berries are known to have disease preventive and anticancer properties. We studied whether supplementing a habitual diet rich in red meat with berries affects cancer biomarkers in a clinical trial with 43 healthy volunteers. We exposed Caco-2 and HCA-7 colon adenocarcinoma cells and HaCaT cells, representing non-cancerous epithelial tissue, in 2D and 3D cultures to faecal water extracted from stool samples collected at baseline and at the end of the 4-wk trial. Expression of markers from the Wnt/β-catenin and PI3K/AKT/mTOR pathways, the cell proliferation marker Ki67, and the apoptosis marker cleaved caspase-3 were detected by Western blotting and immunofluorescence staining methods. In addition, genotoxicity of faecal water and the concentration of N-nitroso compounds in the faeces were analysed. The diet supplemented with berries caused downregulation of β-catenin, PS6 as well as Ki67 signalling in the cells exposed to faecal water. Our results suggest that consuming berries as part of a habitual Western-type diet could lead to less cancerous colon metabolism and possibly lower the risk for colorectal cancer by modulating the central signalling pathways in cancer.

Dietary N-Nitroso Compounds and Risk of Hepatocellular Carcinoma: A USA-Based Study.

N-nitroso compounds (NOCs) are among the most potent dietary carcinogens. N-nitrosodiethylamine (NDEA), N-nitrosodimethylamine (NDMA), and N-nitrosopiperidine (NPIP) are abundant in foods and carcinogenic to the liver. We investigated the relationship between dietary NOCs and HCC risk. In this large, hospital-based, case-control study of 827 pathologically or radiologically confirmed HCC cases and 1,013 controls, NOC intake was calculated by linking food frequency questionnaire-derived dietary data with a comprehensive NOC concentration database. Multivariable-adjusted ORs and 95% CIs of HCC by quartiles of NOC consumption were estimated using logistic regression models, with the lowest quartile as the referent. We further investigated joint effects of consuming the highest quartile of NOCs that were associated with increased HCC risk and hepatitis, diabetes, or alcohol drinking on HCC risk. After adjustment for confounding factors, higher intake of NDEA from plant sources (ORQ4 vs. Q1 =1.58; 95% CI=1.03-2.41), NDMA from plant sources (ORQ4 vs. Q1 =1.54; 95% CI=1.01-2.34), and NPIP (ORQ4 vs. Q1 =2.52; 95% CI=1.62-3.94) was associated with increased HCC risk. No association was observed for nitrate or total NOC intake and HCC risk. Higher consumption of HCC-inducing NOCs and positive hepatitis virus status jointly increased the risk of developing HCC. In conclusion, though some of our findings may indicate the presence of reverse causation owing to lower meat intake among cases with chronic liver diseases before HCC diagnosis, the potent dietary HCC carcinogens, NDEA, NDMA, and NPIP, and their enhanced carcinogenic effects among chronic carriers of hepatitis virus warrant further prospective investigation.

Dietary N-nitroso compounds and risk of pancreatic cancer: results from a large case-control study.

N-nitroso compounds (NOCs) are among the most potent dietary and pancreatic carcinogens. N-nitrosodiethylamine (NDEA) and N-nitrosodimethylamine (NDMA) are the most prevalent NOCs identified in foods. Using a validated and comprehensive N-nitroso database developed to estimate total NOCs and important individual NOCs from food intake, we investigated dietary exposure to NOCs in relation to pancreatic cancer in a large matched case-control study. Self-administered food frequency questionnaires were collected from 957 pathologically confirmed pancreatic ductal adenocarcinoma cases and 938 frequency-matched controls. For each food item, frequency of intake and portion size in grams was multiplied by the estimated NOC concentration from the N-nitroso database. Multiple unconditional logistic regression models were used to estimate the odds ratios (OR) and 95% confidence intervals (CIs) for pancreatic cancer risk by quartiles of NOCs and major food group contributors to NOCs, with the lowest quartile as referent. Following adjustment for confounders, we observed significant positive associations for NDEA (ORQ4 versus Q1 = 2.28, 95% CI = 1.71-3.04, Ptrend < 0.0001) and NDMA from plant sources (ORQ4 versus Q1 = 1.93, 95% CI = 1.42-2.61, Ptrend < 0.0001) with pancreatic cancer. The major food groups related to NDEA and NDMA intakes in this population were fermented cheese, pizza, grains, seafood and beer. No associations of intake of nitrate or total NOCs were observed; nitrite was inversely associated with pancreatic cancer. Although some of our findings probably reflect reverse causation bias due to lower meat intake in cases with latent disease, biologically plausible findings for pancreatic carcinogens, NDEA and NDMA, warrant further prospective investigation.

Diet Composition Is Associated with Endogenous Formation of N-Nitroso Compounds in Obese Men1–3

Endogenous formation of carcinogenic N-nitroso compounds (NOC) occurs in the human gut. Red meat is considered the most important dietary component linked to NOC formation, although nitrate and vitamin C (VitC) also contribute. We previously showed that high-protein weight-loss diets increased fecal NOC and this was enhanced by simultaneous carbohydrate restriction. Although previous studies have focused on the effect of either 1 or 2 dietary components on endogenous NOC formation, no study to date has investigated the combined contribution of various dietary components. The current study therefore assessed the joint impact of several known dietary contributors to the endogenous formation of NOC in obese men. It also aimed to identify further novel contributors and investigate their role in explaining shifts in endogenous formation of NOC. Three dietary trials were conducted in obese men consuming body weight maintenance or weight-loss diets, with NOC measured in fecal samples. Consumption of meat-based weight-loss diets increased (P < 0.001) fecal NOC. Red meat intake was positively correlated with the fecal log NOC concentration (r = 0.60; P < 0.001). Dietary carbohydrate and sugar were negatively correlated with the fecal log NOC concentration (r = -0.66 for both; P < 0.001). Multiple regression analysis identified several dietary components that drive endogenous NOC formation, namely, red meat, nitrate, VitC, total energy, and nonstarch polysaccharides. We present a regression model that predicts endogenous NOC formation in obese men based on their dietary intakes. This model could improve the estimation of endogenous NOC formation, currently used in epidemiological studies into diet and cancer.

N-nitroso compound exposure-associated transcriptomic profiles are indicative of an increased risk for colorectal cancer

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.

Radical Mechanisms in Nitrosamine- and Nitrosamide-Induced Whole-Genome Gene Expression Modulations in Caco-2 Cells

N-nitroso compounds (NOCs) may be implicated in human colon carcinogenesis, but the toxicological mechanisms involved have not been elucidated. Because it was previously demonstrated that nitrosamines and nitrosamides, representing two classes of NOC, induce distinct gene expression effects in colon cells that are particularly related to oxidative stress, we hypothesized that different radical mechanisms are involved. Using electron spin resonance spectroscopy, we investigated the radical-generating properties of genotoxic NOC concentrations in human colon adenocarcinoma cells (Caco-2). Cells were exposed to nitrosamides (N-methyl-N'-nitro-N-nitrosoguanidine and N-methyl-N-nitrosourea) or nitrosamines (N-nitrosodiethylamine, N-nitrosodimethylamine, N-nitrosopiperidine, and N-nitrosopyrrolidine). Nitrosamines caused formation of reactive oxygen species (ROS) and carbon-centered radicals, which was further stimulated in the presence of Caco-2 cells. N-methyl-N-nitrosourea exposure resulted in a small ROS signal, and formation of nitrogen-centered radicals (NCRs), also stimulated by Caco-2 cells. N-methyl-N'-nitro-N-nitrosoguanidine did not cause radical formation at genotoxic concentrations, but at increased exposure levels, both ROS and NCR formation was observed. By associating gene expression patterns with ROS formation, several cellular processes responding to nitrosamine exposure were identified, including apoptosis, cell cycle blockage, DNA repair, and oxidative stress. These findings suggest that following NOC exposure in Caco-2 cells, ROS formation plays an important role in deregulation of gene expression patterns that may be relevant for the process of chemical carcinogenesis in the human colon, in addition to the role of DNA alkylation.

The effect of intra-gastric acidity and flora on the concentration of N-nitroso compounds in the stomach

Correa's hypothesis proposes that gastric carcinogenesis is due to atrophic gastritis and hypochlorhydria which permit gastric bacterial colonization, the reduction of dietary nitrates to nitrites and the formation of potentially carcinogenic N-nitroso compounds (NOCs). To test the hypothesis that omeprazole-induced hypochlorhydria is associated with increased intra-gastric concentrations of nitrate-reducing bacteria (NRB), nitrites and NOCs. Single-blind study in healthy volunteers. Fourteen healthy subjects (seven female, mean age 24 years), free of Helicobacter pylori infection, received a one-week course of placebo followed by a two-week course of omeprazole, 20 mg daily. Fasted gastric samples, aspirated using a sterile double-lumen nasogastric tube at the end of the 1 st week (placebo) and the 2nd and 3rd weeks (omeprazole), were cultured aerobically and anaerobically; gastric pH and intra-gastric concentrations of nitrates, nitrites and NOCs were also determined. After weeks 1, 2 and 3, the intra-gastric concentrations of nitrate-reducing bacteria exceeded 10(5) colony-forming units (c.f.u.)/ml in 3, 7 and 9 subjects, respectively (P > 0.05). A gastric pH greater than 4.0 was associated with increased NRB (P < 0.05); however, neither increased gastric pH nor increased NRB, alone or in combination, was associated with increased intra-gastric concentrations of nitrites or NOCs (P > 0.05). A two-week increase in gastric pH in healthy, H. pylori-negative subjects was associated with increased intra-gastric concentrations of nitrate-reducing bacteria but not of nitrites or N-nitroso compounds. These data suggest that reduced gastric acid secretion is not a necessary precursor to the formation of carcinogenic N-nitroso compounds and that other mechanisms should be invoked to explain gastric carcinogenesis.

N-Nitroso compounds in the diet

N-Nitroso compounds were known almost 40 years ago to be present in food treated with sodium nitrite, which made fish meal hepatotoxic to animals through formation of nitrosodimethylamine (NDMA). Since that time, N-nitroso compounds have been shown in animal experiments to be the most broadly acting and the most potent group of carcinogens. The key role of nitrite and nitrogen oxides in forming N-nitroso compounds by interaction with secondary and tertiary amino compounds has led to the examination worldwide of foods for the presence of N-nitroso compounds, which have been found almost exclusively in those foods containing nitrite or which have become exposed to nitrogen oxides. Among these are cured meats, especially bacon—and especially when cooked; concentrations of 100 μg kg −1 have been found or, more usually, near 10 μg kg −1. This would correspond to consumption of 1 μg of NDMA in a 100-g portion. Much higher concentrations of NDMA (but lower ones of other nitrosamines) have been found in Japanese smoked and cured fish (more than 100 μg kg −1). Beer is one source of NDMA, in which as much as 70 μg l −1 has been reported in some types of German beer, although usual levels are much lower (10 or 5 μg l −1); this could mean a considerable intake for a heavy beer drinker of several liters per day. Levels of nitrosamines have been declining during the past three decades, concurrent with a lowering of the nitrite used in food and greater control of exposure of malt to nitrogen oxides in beer making. There have been declines of N-nitroso compound concentrations in many foods during the past two decades. The small amounts of nitrosamines in food are nonetheless significant because of the possibility—even likelihood—that humans are more sensitive to these carcinogens than are laboratory rodents. Although it is probable that alkylnitrosamides (which induce brain tumors in rodents) are present in cured meats and other potentially nitrosated products in spite of much searching, there has been only limited indirect evidence of their presence.

Fine ParticulateN–Nitroso and Nitrite Organic Compounds in the Atmosphere

Abstract Photochemistry between gas phase organic compounds, oxides of nitrogen, and ozone is expected to result in the formation of nitrogen-containing compounds of potential toxicological importance. Classes of compounds which may form include nitro-, N-nitroso-, nitrate-, and nitrite-substituted organic compounds. Many of these compounds are labile, semivolatile organic compounds in equilibrium between the gas and particulate phases in the atmosphere. The phase distribution of these potentially toxic semivolatile organic compounds can be determined using diffusion denuder sampling technology. The total concentration of N-nitroso compounds in a collected sample is determined using N-nitroso-specific denitrosation reactions followed by detection of the produced nitric oxide with a chemiluminescence detector. Denitrosation chemistry can also be used to determine nitrite compounds. Differentiation between total N-nitroso- and nitrite-containing compounds is accomplished using sulfamic acid as a nitrite-spe...

The Determination of Total Nitrite and N-Nitroso Compounds in Atmospheric Samples

A group-selective method for the determination of total N-nitroso compounds in air samples was developed. N-nitroso compounds in collected samples are selectively decomposed to yield nitric oxide, which is chemiluminescently detected using a thermal energy analyzer (TEA). The detection system is rapid, reproducible and sensitive, with a detection limit of 0.8 pmol. The method requires only 1–3 microliters of sample extract solution and can be used to analyze aqueous solutions. Nitrites interfere with the determination. However, by analyzing samples before and after treatment with sulfamic acid, total nitrite concentration can be measured from the difference in the two determinations. The analytical technique was applied to the analysis of three collocated samples with a quartz filter followed by an XAD-II trap. The average concentration of total N-nitroso compounds collected on the quartz filters was 12.7 ± 2.4 pmol/m3. The concentration of N-nitroso compounds collected by XAD-II was 19.1 ± 3.3 pmol/m3. The total nitrite concentrations were 28.2 ± 3.4 pmol/m3 and 25.5 ± 18.1 pmol/m3 for material collected on the quartz filter and XAD-II, respectively. A fourth collocated sample spiked with diethylamine on the quartz filter gave higher concentrations of N-nitroso compounds in both the filter and the XAD-II bed. This increase indicates that artifact formation of N-nitroso compounds occurred during sampling. It is recommended that these artifacts be avoided in future sample collection by the use of diffusion denuders to remove the gas phase species before the collection of particles for accurate N-nitroso compounds analysis in ambient air.

N-nitroso compounds in two nitrosated food products in Southwest Korea

Gastric cancer is the commonest malignant neoplasm in Southwest Korea. The possibility of carcinogenic dietary factors led to the investigation of exposure to N-nitroso compound precursors among residents of the City of Chonju and of two outlying rural townships in North Cholla Province. Two traditional and widely consumed home-prepared food products, salted pickled cabbage ( kimchi) and salted seafood sauce ( chut-kal) were analysed (a) for nitrite, nitrate, total secondary amines and pH in these food products prior to nitrite incubation and (b) for volatile nitrosamines and total N-nitroso compounds before and after incubation with nitrite in simulated human stomach conditions. Nitrate levels were significantly higher in kimchi (median 1550 mg/kg) than in chut-kal (median 140 mg/kg) ( P < 0.001). Secondary amine levels in non-nitrosated samples of kimchi (median 5.5 mg/kg) were significantly lower than secondary amine levels in non-nitrosated chut-kal (median 56 mg/kg) ( P = < 0.001). Analyses of nitrite-incubated kimchi revealed high levels of total N-nitroso compounds (median 1173 μg/kg); the increase with nitrosation was significant ( P = 0.001). The concentration of N-nitroso compounds in nitrite-incubated kimchi was significantly greater than that found in nitrite-incubated chut-kal ( P = 0.015). The combination of high levels of nitrate in the kimchi, the demonstration of high levels of total N-nitroso compounds in this food after nitrosation, and the volume of kimchi consumed in the traditional diet suggest that salted pickled cabbage may play a role in gastric carcinogenesis in Southwest Korea.

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.

Instability of N-nitroso compounds in gastric juice and preliminary results from analyses of fresh samples by using an improved analytical method

Using an improved method for determination of total N-nitroso compounds (NOC), we examined the stability of those compounds in fresh gastric juice samples during storage and the effects of the addition of 2% sulphamic acid on NOC concentration in 212 samples. The NOC levels in fresh samples decreased very rapidly at -20 degrees C, especially during the first 24 hours of storage (P < 0.01), and nitrite concentration also showed a decreasing trend during storage. The addition of sulphamic acid significantly reduced NOC levels from 1.97 +/- 0.21 to 1.10 +/- 0.12 mumol/l (mean +/- SE, P < 0.01), especially in samples of initially high pH. However, in some individual samples (16.5%) the NOC levels actually increased by 14.1% (P < 0.01). The results from analysis of NOC in 212 fresh samples in relation to pH demonstrated two significantly higher peaks of NOC concentrations at intragastric pH ranges 1.1 to 2.99 (P < 0.05) and 6.0 to 7.9 (P < 0.01). There was a significant relationship between nitrite level and intragastric pH (r = 0.480, P < 0.01), the nitrite concentration increasing dramatically when the pH exceeded 6.0. The present study suggest that a major proportion of the unidentified NOC formed through intragastric nitrosation is labile NOC; if the true concentration of NOC is to be determined it is therefore essential to analyse fresh gastric juice samples directly after collection and without pretreatment. It will also be necessary to characterize those labile NOC in order to study further the mechanism of endogenous N-nitrosation in man and its relation to human carcinogenesis.

Primary Adenocarcinoma Arising in the Jejunal Limb of a Roux-en-Y Esophago-jejunostomy: A Case Report

Adenocarcinoma of the small bowel is uncommon. Due to the paucity and lack of specificity of symptoms, patients are usually seen late in the course of their illness when curative therapy, mainly represented by wide surgical resection, is unlikely. The authors report a case of primary adenocarcinoma arising in the jejunal limb transposed for a Roux-en-Y esophagojejunostomy reconstruction eight years after a total gastrectomy performed for an advanced gastric cancer (pT2N1M0), with mixed histological pattern (tubular-mucinous) and negative CEA staining of cancer cells. Evidence for excluding the possibility of a recurrence of the primitive gastric cancer was based on the different histologic pattern, positive CEA staining of cancer cells and other features of the second neoplasm. Early diagnosis of the neoplasm was made possible by its favorable anatomic location and the early onset of symptoms, which prompted effective surgical therapy (wide resection). The authors, furthermore, analyze and discuss the possible pathogenesis of the neoplasm, based on the evidence of slow jejunal limb emptying, elevated concentration of N-Nitroso compounds and contamination by N-nitrosating bacteria in the jejunal limb juice, suggesting that each of these factors could have had a role to play in the development of the jejunal malignancy.

N-nitroso compounds in fresh gastric juice and their relation to intragastric pH and nitrite employing an improved analytical method.

In order to examine further the relationship between intragastric N-nitrosation, gastric pH and nitrite, 457 fresh, fasting gastric juice samples were analysed for total N-nitroso compounds (NOC) and nitrite concentrations using a recently described improved assay method. Nitrite in log values was linearly related to intragastric pH (r = 0.887, P < 0.01) with a regression equation log[nitrite] (mumol/l) = 0.489 x pH - 2.209. Significantly higher NOC concentrations were found at intragastric pH ranges of 1.13-2.99 (mean +/- SE: 1.45 +/- 0.17 mumol/l, P < 0.05) and 6.00-8.42 (3.57 +/- 0.33 mumol/l, P < 0.01) compared with that at pH 3.00-5.99 (1.02 +/- 0.12 mumol/l). NOC concentration was significantly related to log nitrite concentration at both the low pH range 1.13-4.99 (r = 0.169, P < 0.01) and the high pH range 5.00-8.42 (r = 0.450, P < 0.01). The results in the present study confirm that both acid-catalysed N-nitrosation and biologically-catalysed N-nitrosation occur in the human stomach. However, great variations in nitrite and NOC concentrations were observed in both low and high pH samples, indicating that, as expected, both the acid-catalysed N-nitrosation and biologically-catalysed N-nitrosation processes are markedly affected by factors other than intragastric pH and nitrite.

Determination of Total N-Nitroso Compounds in Biological Fluids By Photolytic Denitrosation and Thermal Energy Analysis

ABSTRACT Quantification of total N-nitroso compounds (NOC) in urine and gastric juice is achieved by combining photolytic denitrosation with thermal energy analysis. The minimum mass detection limit of the technique is about 10 pmole No. Urine samples with NOC concentration as low as 200 nmolL−1 can be analyzed with a precision of approximately 5-10%. The minimum concentration detection limit for gastric juice samples is about 1 μmolL−1. Except for nitrite, no interference is observed from nitrogenous compounds present in urine and gastric juice. The nitrite interference is effectively eliminated with sulphamic acid.

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 μmol/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: ⩽0.01–8.0 μmol/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 ⩽ 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 μmol/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.

The occurrence of N-nitroso compounds in kiwam tobacco

The concentrations of tobacco-specific nitrosamines (TSNA), volatile and non-volatile N-nitroso compounds in kiwam, a fermented Indian tobacco product are presented. Total identified N-nitroso compound concentrations in kiwam ranged from 6.19 to 25.4 mg/kg fresh weight tobacco, the concentration range of TSNA was 5.43-22.2 mg/kg tobacco which accounted for 67-87% of the total identified N-nitroso compound burden. The high concentrations of TSNA found in kiwam tobacco may present a considerable exposure source to carcinogenic N-nitroso compounds for people using this type of tobacco.

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.