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Background: Both N-nitroso compounds and colonization with Helicobacter pylori represent known risk-factors for the development of gastric cancer. Endogenous formation of N-nitroso compounds is thought to occur predominantly in acidic environments such as the stomach. At neutral pH, bacteria can catalyze the formation of N-nitroso compounds. Based on experiments with a noncarcinogenic N-nitroso compound as end product, and using only a single H. pylori strain, it was recently reported that H. pylori only displays a low nitrosation capacity. As H. pylori is a highly diverse bacterial species, it is reasonable to question the generality of this finding. In this study, several genetically distinct H. pylori strains are tested for their capacity to form carcinogenic N-nitrosamines., Materials and Methods: Bacteria were grown in the presence of 0-1000 microM morpholine and nitrite (in a 1 : 1 molar ratio), at pH 7, 5 and 3., Results: Incubation of Neisseria cinerea (positive control) with 500 microM morpholine and 500 microM nitrite, resulted in a significant increase in formation of N-nitrosomorpholine, but there was no significant induction of N-nitrosomorpholine formation by any of the H. pylori strains, at any of the three pH conditions., Conclusion: H. pylori does not induce formation of the carcinogenic N-nitrosomorpholine in vitro. The previously reported weak nitrosation capacity of H. pylori is not sufficient to nitrosate the more difficulty nitrosatable morpholine. This probably also holds true for other secondary amines. These results imply that the increased incidence of gastric cancer formation that is associated with gastric colonization by H. pylori is unlikely to result from the direct induced formation of carcinogenic nitrosamines by H. pylori. However, this has to be further confirmed in in vivo studies.

Background & Aims: This study evaluated the effect of long-term gastric acid suppressive therapy with omeprazole on intragastric levels of carcinogenic N-nitrosamines and related parameters., Methods: Forty-five patients on long-term omeprazole medication (mean, 35 months) and 13 healthy subjects without medication participated. Volatile N-nitrosamines were determined in gastric juice and urine. Intragastric pH, nitrite, nitrate, and H. pylori status were determined. DNA isolated from gastric biopsy specimens was analyzed for precarcinogenic alkyl-DNA adducts., Results: The intragastric pH in patients was significantly higher compared with controls (P = 0.0001). Gastric nitrite levels in patients were nonsignificantly higher. There was no difference in total levels of intragastric volatile N-nitrosamines between patients and controls, however, urinary N-nitrosodimethylamine excretion was higher in patients (P = 0.001). On omeprazole, Helicobacter pylori-positive vs. -negative patients had a nonsignificantly higher intragastric nitrite level and higher urinary N-nitrosodimethylamine excretion. No alkyl-DNA adducts could be detected in gastric epithelium., Conclusions: Increased intragastric pH caused by long-term treatment with omeprazole does not result in increased intragastric levels of nitrite and volatile N-nitrosamines. The significantly higher urinary N-nitrosamine excretion implies the risk of increased endogenous formation of N-nitrosamines during long-term omeprazole treatment. This risk may be higher in H. pylori-positive patients.

The thermal energy analyser (TEA) is considered to be the gold standard for the determination of nitrosamines. However, since many laboratories cannot justify the use of such a very specific detection system, alternative detection methods are useful. While standard gas chromatography (GC) detectors lack the selectivity of the TEA detector, mass spectrometry (MS) seems to be the method of choice to combine GC separation with mass selective detection. Moreover, the detection limits of the GC-MS assay in general use are about 4 times lower than those of the GC-TEA assay. A comparison of GC-MS and GC-TEA data on N-nitrosodimethylamine determinations showed a strong correlation between the two assays (R2 = 0.86), demonstrating the exchangeability of these methods.

Urinary excretion of volatile nitrosamines was assessed in 59 non-smokers living in a rural county of Québec, Canada. Water and food intakes were measured by means of a 24-hour recall. Nitrates were analyzed in the tap water of all participants (geometric mean=2.0 mg nitrate-N/L) and dietary intakes of nitrate and vitamins C and E were estimated via a validated Canadian food database. Urine was collected over the same 24-hour period and analyzed for nitrates by hydrazine reduction and for volatile nitrosamines by gas-chromatography/mass spectrometry. N-Nitrosopiperidine (NPIP) was found in urine samples from 52 of the 59 subjects. Geometric mean of NPIP urinary excretion was 67 ng/day and maximum value was 1045 ng/day. No other volatile nitrosamine was detected. There was a correlation between urinary nitrate excretion and total nitrate intake (r=0.71, P < 0.001). However, no relationship was found between urinary NPIP excretion and either nitrate excretion, dietary or water nitrate intakes. NPIP excretion was significantly correlated to coffee intake (r=0.40, P=0.002) and this relation was not modified by vitamin intake. We conclude that nitrate intake is not related to nitrosamine excretion in this rural population. The influence of coffee consumption on NPIP excretion deserves further attention.

Many constituents present in the human diet may inhibit endogenous formation of N-nitroso compounds (NOC). Studies with human volunteers showed inhibiting effects of intake of ascorbic acid and green tea consumption on nitrosation using the N-nitrosoproline test. The aim of the present study was to evaluate the effects of ascorbic acid and green tea on urinary excretion of carcinogenic N-nitrosodimethylamine (NDMA) and N-nitrosopiperidine (NPIP) in humans. Twenty-five healthy female volunteers consumed a fish meal rich in amines as nitrosatable precursors in combination with intake of nitrate-containing drinking water at the Acceptable Daily Intake level during 7 consecutive days. During 1 week before and after nitrate intake a diet low in nitrate was consumed. Using the same protocol, the effect of two different doses of ascorbic acid (250 mg and 1 g/day) and two different doses of green tea (2 g and 4 g/day) on formation of NDMA and NPIP was studied. Mean nitrate excretion in urine significantly increased from control (76+/-24) to 167+/-25 mg/24 h. Intake of nitrate and fish resulted in a significant increase in mean urinary excretion of NDMA compared with the control weeks: 871+/-430 and 640+/-277 ng/24 h during days 1-3 and 4-7, respectively, compared with 385+/-196 ng/24 h (p<0.0002). Excretion of NPIP in urine was not related to nitrate intake and composition of the diet. Intake of 250 mg and 1 g of ascorbic acid per day resulted in a significant decrease in urinary NDMA excretion during days 4-7 (p=0.0001), but not during days 1-3. Also, consumption of four cups of green tea per day (2 g) significantly decreased excretion of NDMA during days 4-7 (p=0.0035), but not during days 1-3. Surprisingly, consumption of eight cups of green tea per day (4 g) significantly increased NDMA excretion during days 4-7 (p=0.0001), again not during days 1-3. This increase is probably a result of catalytic effects of tea polyphenols on nitrosation, or of another, yet unknown, mechanism. These results suggest that intake of ascorbic acid and moderate consumption of green tea can reduce endogenous NDMA formation.

Formation of nitrite from ingested nitrate can result in several adverse health effects and implies a genotoxic risk as a consequence of endogenous formation of carcinogenic N-nitroso compounds. We studied the formation of volatile N-nitrosamines after intake of nitrate at the acceptable daily intake (ADI) level in combination with a fish meal rich in amines as nitrosatable precursors. Twenty-five volunteers consumed this meal during 7 consecutive days; a diet low in nitrate was consumed during 1 week before and 1 week after the test week. Nitrate intake at the ADI level resulted in a significant rise in mean salivary nitrate and nitrite concentrations. Mean urinary nitrate excretion increased from 76 mg/24 hr in the first control week to 194 and 165 mg/24 hr in the test week, followed by a decline to 77 mg/24 hr in the second control week. The urine samples were analyzed for volatile N-nitrosamines, and both N-nitrosodimethylamine (NDMA) and N-nitrosopiperidine (NPIP) were detected in the samples. Mean urinary NDMA excretion significantly increased from 287 ng/24 hr in the control week to 871 and 640 ng/24 hr in the test week and declined to 383 ng/24 hr in the second control week. Excretion of NPIP was not directly related to the nitrate intake and composition of the diet. Nitrate excretion and NDMA excretion were significantly correlated, as well as salivary nitrate and nitrite concentration and NDMA excretion. We conclude that nitrate intake at the ADI level in combination with a fish meal containing nitrosatable precursors increases NDMA excretion in urine and thus demonstrates increased formation of carcinogenic N-nitrosamines.

Gastric juice samples of 71 patients undergoing upper gastrointestinal endoscopy were collected as well as saliva samples from 40 of these patients. Age, sex, endoscopic diagnosis and medication were recorded. The gastric juice samples were analyzed for the presence and quantity of individual volatile N-nitrosamines, which were detected by gas chromatography-mass spectrometry, without prior derivatization. The samples were screened for eight nitrosamines, i.e. N-nitrosodimethylamine, N-nitrosoethylmethylamine, N-nitrosodiethylamine, N-nitrosodi-n-propylamine, N-nitrosodi-n-butylamine, N-nitrosopyrrolidine, N-nitrosopiperidine and N-nitrosomorpholine. The pH of the fresh gastric juice as well as nitrate and nitrite levels of gastric juice and saliva were determined. The mean total level of volatile N-nitrosamines in gastric juice was found to be 4.84 nmol/l (range 0-17.7 nmol/l). The main N-nitrosamines found were N-nitrosodiethylamine (mean concentration 3.1 nmol/l), N-nitrosodimethylamine (mean concentration 0.90 nmol/l) and N-nitrosopyrrolidine (mean concentration 0.38 nmol/l). Significant correlations between mean intragastric pH values and mean N-nitrosodi-n-butylamine level (P = 0.005) and total volatile N-nitrosamine contents (P = 0.009) were observed.

We studied the formation of carcinogenic nitrosamines during consumption of food rich in nitrate and amines, and its possible inhibition by use of an antibacterial mouthwash. Twelve volunteers were fed a diet containing the high-nitrate vegetables lettuce or spinach during two periods of four consecutive days, in combination with fish products containing high levels of amines as nitrosatable precursors. During the two periods, the subjects used an antibacterial mouthwash containing chlorhexidine or a control mouthwash without antibacterial activity. Twenty-four-hour urine samples were collected after consumption of the meals, and saliva samples were collected 1 h after each meal. The nitrate and nitrite contents of the urine and saliva samples were determined by spectrophotometry (for nitrite) and HPLC (for nitrate). The concentrations of volatile nitrosamines in the urine samples were determined by gas chromatography-mass spectrometry. Significant increases in mean urinary nitrate levels (from 59 to 135 mg/24 h) and in mean salivary nitrate levels (from 10 to 56 microg/ml) and salivary nitrite levels (from 2 to 11 microg/ml) were observed during the consumption of food rich in nitrate and amines, as well as a significant increase in the mean urinary excretion of total examined volatile nitrosamines (from 2 to 7 nmol/24 h) and of N-nitrosodimethylamine (from 1.2 to 2.9 nmol/24 h). Use of the antibacterial mouthwash resulted in a decrease in mean salivary nitrite levels from 16 to 3 microg/ml and a decrease in mean urinary excretion of N-nitrosomorpholine (from 7.0 to 0.3 nmol/24 h). For the whole data set, significant correlations were observed between nitrate intake in food and urinary nitrate (p = 0.01; r2 = 0.07) and between urinary nitrate and urinary N-nitrosodimethylamine (p = 0.002; r2 = 0.11). In conclusion, consumption of a diet rich in nitrate and amines increases the risk of formation of carcinogenic nitrosamines. Use of an antibacterial mouthwash containing chlorhexidine can result in inhibition of nitrosamine formation.