Formation Of N-nitroso Compounds Research Articles

Reactions of nitrate and nitrite in foods with special reference to the determination of N-nitroso compounds.

The stabilities of nitrate and nitrite in food systems and their reactions in such matrices are reviewed. Particular reference is made to reactions with haem proteins, smoke components and amines in foods, and the chemistry of formation of N-nitroso compounds from food components is discussed. Finally, the methodology available for determination of both volatile and non-volatile N-nitroso compounds is addressed.

The effects of Chinese tea on the occurrence of esophageal tumors induced by N-nitrosomethylbenzylamine in rats

Background. Based on previous studies on the blocking effect of Chinese tea in the formation of N-nitroso compounds in rats and humans, experiments were carried out to study the effects of Chinese tea on the occurrence of esophageal tumors induced by N-nitrosomethylbenzylamine (NMBzA) in rats. Methods. In the first experiment, rats were given two precursors of NMBzA, i.e., sodium nitrite and methyl benzylamine, p.o. After 12 weeks, the incidence of esophageal tumors was 95%. However, in the five groups of tea (green tea, jasmine tea, black tea, and oolong tea)-treated rats, the incidences were only 5–19%. In the second experiment, preformed NMBzA was administered to rats. Results. The incidences of esophageal tumors in the five tea-treated groups were 42–67%, while in the positive control group, without tea, the incidence was 90%. Histopathological examination showed the same protective effects of tea treatment. In a separate study, a significant reduction of O 6-methylguanine (MeG) and the ratio of O 6-MeG to N 7-MeG was observed in rats treated with oolong tea and jasmine tea.

Nitrosamine Excretion in Patients with Continent Ileal Reservoirs for Urinary Diversion

Adenocarcinomas are a recognized complication following ureterosigmoidostomy for which the endogenous formation of N-nitroso compounds may be a risk factor. As an alternative means of urinary diversion, the continent ileal reservoir has recently been developed. Microbiological and chemical investigations on the urine of patients with an ileal reservoir showed the presence of bacteria, nitrate, nitrite and N-nitrosamines formed endogenously in the ileal pouch. The role of nitrosamines in carcinogenesis in these patients as a late stage complication resulting from the use of a continent ileal reservoir is discussed.

Review of the occurrence and formation of non-volatile N-nitroso compounds in foods.

A review of the literature published prior to July 1991 covers the occurrence and formation of non-volatile N-nitrosamines occurring in foods and beverages. The presence of identified volatile and non-volatile N-nitrosamines accounts for less than 10% of the total apparent N-nitrosamine concentration. N-Nitrosoproline and N-nitrosothiazolidine-4-carboxylic acid are the most commonly identified non-volatile N-nitrosamines in the diet. Non-volatile N-nitrosamines account for 12 of the 21 currently identified N-nitroso compounds in foods and beverages.

N-nitrosamine and mutagenicity formation in Chinese salted fish after digestion.

Salted and dried fish (Nemipterus virgatus), acquired from Hong Kong, was treated with 0.43-110 mM nitrite during in vitro digestion using gastric enzymes and the volatile N-nitrosamine content and mutagenicity on Salmonella typhimurium TA100 assayed without concentration. N-Nitrosodimethylamine (NDMA; the only nitrosamine detected) formation was second order in nitrite concentration. When 10 g of fish was treated with 6.96 mM nitrite, 394 nM NDMA was formed. Thiocyanate was catalytic for NDMA formation at nitrite concentration greater than 0.87 mM and when the ratio of thiocyanate to nitrite was greater than 1. Approximately a 50% inhibition in NDMA formation by ascorbic acid was seen when the ratio of ascorbate to nitrite was approximately 2 or greater and the nitrite concentration was 1.74 mM. Mutagenicity increased with increasing nitrite concentration but the addition of thiocyanate did not increase mutagenicity over nitrite alone. Ascorbate increased mutagenicity even though NDMA formation was inhibited. Even at nitrite concentrations greater than 100-fold higher than expected in vivo, there was insufficient NDMA formed to account for the observed mutagenicity. These data do not exclude the possibility that the observed mutagenicity was due to non-volatile N-nitroso compounds, however, this possibility seems unlikely given the effects of ascorbate and thiocyanate which would be expected to inhibit and enhance non-volatile N-nitroso compound formation.

Nitrate contamination of drinking water: evaluation of genotoxic risk in human populations.

Nitrate contamination of drinking water implies a genotoxic risk to man due to the endogenous formation of carcinogenic N-nitroso compounds from nitrate-derived nitrite. Thus far, epidemiological studies have presented conflicting results on the relation of drinking water nitrate levels with gastric cancer incidence. This uncertainty becomes of relevance in view of the steadily increasing nitrate levels in regular drinking water supplies. In an attempt to apply genetic biomarker analysis to improve the basis for risk assessment with respect to drinking water nitrate contamination, this study evaluates peripheral lymphocyte chromosomal damage in human populations exposed to low, medium, and high drinking water nitrate levels, the latter being present in private water wells. It is shown that nitrate contamination of drinking water causes dose-dependent increases in nitrate body load as monitored by 24-hr urinary nitrate excretion in female volunteers, but this appears not to be associated with peripheral lymphocyte sister chromatid exchange frequencies.

Role of glucosinolates in the formation of N-nitroso compounds

Since thiocyanate ion is able to catalyse the nitrosation reaction, the breakdown products of glucosinolates were further tested for their ability to catalyse the nitrosation rate of proline. Because the low pH of the stomach may result in the chemical breakdown of glucosinolates, the hydrolysis of glucosinolates (sinigrin, gluconapin, progoitrin, glucotropaeolin, sinalbin gluconasturtiin, glucobrassicin, and 4-hydroxyglucobrassicin) were also examined.

Temporal influence of ascorbic acid dose on the endogenous formation of N-nitrosoproline and N-nitrosothiazolidine-4-carboxylic acid in humans

The temporal effect of ascorbic acid intake on the endogenous formation of N-nitrosoproline and N-nitrosothiazolidine-4-carboxylic acid was determined over 15 days in 10 ascorbate depleted men consuming a low nitrate and ascorbic acid (<23 μmol) diet. N-nitrosoproline precursors were given as follows: days 3–15, nitrate (5.24 mmol); days 2–15, l-proline (4.35 mmol) 30 min following the nitrate; days 5–11, ascorbic acid (2.62 mmol) 6, 5, 4, 3, 2, 1, and 0.5 hr before the proline, concurrent with the proline on day 12, and 0.5, 1 and 2 hr following the proline on days 13–15. Urine was analyzed for N-nitrosoamino and ascorbic acids. Gastric pH and residence time were measured on days 3–4 concurrently with the proline challenge. Excretion of N-nitrosoproline on nitrate and proline only days was 42.2 ± 16.0 nmol. Significant ( P < 0.05) inhibition was observed when ascorbic acid was given 5 hr before proline (29.1 ± 10.5 nmol). These data suggest that the ascorbic acid content of the stomach due to secretion is sufficient to partially inhibit gastric nitrosation. Diets high in ascorbic acid could reduce gastric formation of N-nitroso compounds even if ascorbic acid is not consumed at the same time as the precursors. No correlation between gastric pH and gastric residence time with N-nitrosoproline and N-nitrosothiazolidine-4-carboxylic acid excretion was observed over the pH range observed. Several subjects consistently excreted high or low levels of N-nitrosoproline or N-nitrosothiazolidine-4-carboxylic acid compared to the mean, regardless of the ascorbic acid intake. This suggests that physiological differences between subjects may account for their endogenous nitrosation capacity.

Endogenous nitrosation of L‐proline by dietary‐derived nitrate

Two studies were conducted to assess the availability of normal dietary sources of nitrate for endogenous nitrosation of an amino acid substrate, L-proline, and to investigate the potential for dietary ascorbic acid to inhibit such nitrosation. In the first study, 16 subjects consumed a salad meal (containing about 172 mg nitrate) with and without a loading dose of proline. A significant increase (10.8 compared with 2.7 micrograms/24 hrs, p less than 0.0001) in mean urinary N-nitrosoproline (NPRO) excretion following meal plus substrate ingestion was indicative of intragastric proline nitrosation by meal-derived nitrate. In the second study (19 different subjects), the mean urinary NPRO level was significantly decreased (15.8 micrograms compared with 28.4, p = 0.022) by inclusion of ascorbic acid sources in the meal. This demonstrated inhibition of proline nitrosation by dietary sources of the vitamin. Large interindividual variation in nitrosating ability was apparent that was associated with variation in meal nitrate to salivary nitrite conversion. Although meals containing fresh vegetables, as tested here, could provide sufficient nitrate to result in endogenous formation of N-nitroso compounds, there will be considerable inter- and intraindividual variation in the extent of this process.

Tobacco Smoking, Chewing, and Snuff Dipping: Factors Contributing to the Endogenous Formation of N-Nitroso Compounds

(1991). Tobacco Smoking, Chewing, and Snuff Dipping: Factors Contributing to the Endogenous Formation of N-Nitroso Compounds. Critical Reviews in Toxicology: Vol. 21, No. 4, pp. 243-253.

Increased urinary nitrosamine excretion in paraplegic patients

An increased risk of bladder cancer is a recognized complication in spine-injured patients undergoing long-term urethral catheterization to preserve renal function. Aerobic cultures from 28 of 30 paraplegic patients showed complex bacterial flora containing nitrate-reducing organisms (Escherichia coli, Proteus and Klebsiella spp.). Urine samples from 29 paraplegic patients were also found to contain volatile nitrosamines. Mean N-nitrosamine excretion levels were 0.65 +/- 0.69 micrograms/day N-nitrosodimethylamine, 0.25 +/- 0.44 micrograms/day N-nitrosopiperidine and 0.39 +/- 0.50 micrograms/day N-nitrosopyrrolidine. A mean urinary nitrite excretion of 10.4 +/- 13.2 mg/day was found in 24 out of 30 paraplegic patients. In the sterile urine of control volunteers (medical staff attending the paraplegic patients and in-patients from other wards of the hospital), no urinary excretion of volatile N-nitrosamines and nitrite was found. The results clearly demonstrate a bacterially mediated in vivo formation of N-nitroso compounds in the urinary tracts of paraplegic patients which may be an important etiological risk factor for bladder cancer in this patient group.

Gastric juice ascorbic acid: effects of disease and implications for gastric carcinogenesis

N-nitroso compounds (NOC) are strongly implicated in the causation of cancer of the stomach and it has been suggested that ascorbic acid might reduce the risk of gastric cancer by preventing their formation within gastric juice. However, until recently there have been no measurements of gastric juice ascorbic acid concentrations. We have measured both gastric juice ascorbic and total vitamin C (ascorbic acid and dehydroascorbic acid). Our findings suggest that ascorbic acid is secreted into the gastric lumen so that gastric juice concentrations are often greater than those in plasma. Gastric pathology affects this secretion, leading to values in gastric juice that are lower than plasma levels. Stimulation of gastric secretion does not raise vitamin C concentrations in individuals whose values are initially low. The role of ascorbic acid in preventing formation of NOC and protecting against gastric cancer is discussed in the light of these findings.

Nitrite from inflammatory cells—A cancer risk factor in ulcerative colitis?

Elevated levels of luminal nitrite and a lowered luminal pH were found in 77 percent of patients with acute ulcerative colitis. No luminal nitrite was found in healthy control subjects. Nitrites are a secretory product of activated macrophages and neutrophils of the lamina propria, whereas the lowered luminal pH is due to diminished bicarbonate formation by impaired colonocytes. A hypothesis is put forward that nitrites, lowered pH, and bacterial amines are conducive to formation of carcinogenic n-nitroso compounds, which reflect a cancer risk in patients with ulcerative colitis dependent on the type and extent of inflammatory cell activation as well as metabolic impairment of colonic epithelial cells.

Occurrence of non‐gastric cancer in the digestive tract after remote partial gastrectomy: Analysis of an Amsterdam cohort

If peptic ulcer surgery favors the formation of carcinogenic N-nitroso compounds in the gastric remnant, an increased risk of cancer at sites in the gastrointestinal tract distant from the stomach might be predicted. To estimate the risk of carcinomas in the digestive tract, other than the stomach, occurring after partial gastrectomy, we analyzed an Amsterdam cohort of 2,633 post-gastrectomy patients operated on for benign disease between 1931 and 1960. Mortality in the study population was compared with the general Dutch population through person-year analysis. An excess mortality of biliary tract cancer (O/E:2.64; CL:1.32-4.72; p less than 0.01) and pancreatic cancer (O/E:1.65; CL:1.06-2.44; p less than 0.05) was found in males more than 5 years after surgery; females showed only an increased risk of pancreatic cancer in the first 5 years postoperatively (O/E:15.33; CL:1.85-55.43; p less than 0.01), probably due to misdiagnosis. All other non-gastric sites of the digestive tract carried no increased risk for cancer. In males, mortality due to colorectal cancer more than 5 years post-operatively was significantly decreased (O/E:0.58; CL 0.34-0.92; p less than 0.01). The excess mortality of biliary-tract and pancreatic cancer in males, which increases with the duration of post-operative interval, is consistent with a dose-response phenomenon. This study therefore supports the hypothesis that carcinogens are not only locally activated in the gastric remnant, but are hepatically excreted and initiate cancer in the biliary tree and pancreatic duct. Further exploration of this mechanism of carcinogenesis is warranted, since it may also explain the pathogenesis of pancreatic and biliary cancers in patients without gastrectomy.

Several known indole compounds are not important precursors of direct mutagenic N-nitroso compounds in green cabbage

In this study we investigated the role of indole-3-acetonitrile, indole-3-carbinol, indole and tryptophan in the formation of N-nitroso compounds in green cabbage extracts. Green cabbage extracts were separated by gel permeation chromatography. Fractions were treated with nitrite, tested for mutagenicity and analysed for total N-nitroso. Fractions in which spiked indole-3-acetonitrile, indole-3-carnibol, indole and tryptophan eluted appeared to be low in mutagenic activity and contained relatively small amounts of N-nitroso compounds. To detect indole compounds other than the ones used in the gel permeation chromatography experiments, high-performance liquid chromatography and gas chromatography-mass spectrometry analyses were performed of green cabbage extracts. Indole-3-carboxaldehyde was found to be the most commonly occuring indole compound, but it did not show direct mutagenic activity upon nitrite treatment. Indole-3-acetonitrile was the second most common compound; although it was mutagenic after nitrite treatment, its contribution to the mutagenicity of nitrite-treated green cabbage was roughly estimated to be only 2%. No other indole compounds were detected. From this study we conclude that neither the tested indole compounds nor indole-3-carboxaldehyde play a significant role in the formation of direct mutagenic N-nitroso compounds in nitrate-treated green cabbage extracts.

Marked formation of thiazolidine-4-carboxylic acid, an effective nitrite trapping agent in vivo, on boiling of dried shiitake mushroom (Lentinus edodes)

Thiazolidine-4-carboxylic acid (TCA, thioproline) is a condensation product of cysteine and formaldehyde. TCA is an effective nitrite trapping agent in the human body and may block endogenous formation of carcinogenic N-nitroso compounds. In this study, was investigated the levels of TCA in five edible mushrooms. Dried samples soaked in water contain a specific system for generating formaldehyde via the formation of lenthionine from lentinic acid

The effect of dietary nitrate on nitrate and nitrite excretion in man

Dietary nitrate and nitrite may affect colonic pathophysiology. These anions influence fermentation, and nitrite has been shown to augment sodium absorption by the colon and participate in the formation of N-nitroso compounds. There is, however, no general agreement as to how much dietary nitrate and nitrite reaches the colon. To help resolve this question, balance studies were performed on six healthy ileostomy subjects who were given diets that varied in nitrate content from 0.83 to 5.20 mmol/d. Nitrate and nitrite excretion in ileal effluent and urine were measured by anion-exchange chromatography with conductivity detection. There was no significant nitrite in the diets, urine or ileal effluent. Dietary nitrate was largely excreted in urine (1.31-4.25 mmol/d). The urinary excretion findings indicated net synthesis of nitrate at low dietary intakes and net catabolism of nitrate at high intakes. Nitrate losses in ileal effluent were very low (0.03-0.05 mmol/d, 0.03-0.06 mmol/kg) and unrelated to intake for all the diets. It is concluded that dietary nitrate and nitrite do not enter the colon from the small intestine in amounts that would affect fermentation and mucosal metabolism in man. The possibility of significant amounts of nitrate reaching the colon via blood in normal subjects has not been excluded.

The role of nitrate, nitrite and N-nitrosamines in carcinogenesis of colon tumours following ureterosigmoidostomy

Urinary diversion in both a rat model for ureterosigmoidostomy and in ureterosigmoidostomy patients result in an increased incidence of colon tumours. Bacterial and chemical investigations on feces-urine mixtures from both the rat model and ureterosigmoidostomy patients showed the presence of a complex nitrate-reducing bacterial flora in both rats and humans. This bacterial flora actively reduced urinary nitrate to nitrite in humans and increased the endogenous formation of N-nitroso compounds. No evidence of urinary nitrate reduction and increased nitrosamine formation in the rectosigmoid of rats was found. The results support the N-nitrosamine theory of carcinogenesis of the colon following ureterosigmoidostomy in humans, but not in rats. As the rat model induces colon carcinomas, factors other than the increased endogenous formation of N-nitroso compounds in the rectosigmoid may contribute to the initiation of colon carcinomas following ureterosigmoidostomy.

Gastric nitrite processing in the surgically altered maximal and minimal bile reflux ferret model

Ingested nitrate and nitrite have been shown to contribute to endogenous, N-nitroso compound formation in man and experimental animals. N-nitroso compounds have long been suspected of contributing to higher levels of gastric cancer in various populations. Reconstructive gastric surgery to treat ulcers is accompanied by a change in bile reflux, gastritis and an increased incidence of gastric cancer in humans. To evaluate possible connections between gastric nitrite processing, reconstructive surgery and gastric cancer, the surgically altered domestic ferret, Mustela putorius furo, was used as an experimental model. The aim of the study was to determine if surgery would alter the stomach in a way which would increase gastric nitrite concentration, and thereby enhance the likelihood of gastric N-nitroso compound formation. Three groups of ferrets, one control group (n = 6) and two groups of surgically altered ferrets, one to simulate maximal bile reflux (MABR, n = 6), and the other to model minimal bile reflux (MIBR, n = 7), were studied. Each group's response to an exogenously administered dose of sodium nitrite did not differ significantly with respect to rate of gastric nitrite absorption, with half-lives in the 13-min range. Permeability of gastric mucosa to nitrite did not differ between controls and MIBR ferrets. Mean doubling time of gastric nitrate appeared slowed in surgically altered ferrets. Mean rate of gastric emptying was the same in the three groups, but appeared delayed initially in MIBR ferrets. Thiocyanate concentrations, pH and HCl secretion, all parameters which have been shown to affect gastric nitrite processing, did not differ significantly between groups. Gastric mucosal endoscopic biopsies obtained at 6-month intervals showed no clear difference in degree of mucosal inflammation and/or dysplasia in the three groups. These findings indicate that gastric mucosal neoplasia has not occurred in this model and that changes in parameters favoring gastric N-nitrosation, even if relevant to the disease process, are not apparent at this time.

INFLUENCE OF NITRATE AND BACTERIAL CONTAMINATION ON THE FORMATION OF APPARENT TOTAL N-NITROSO COMPOUNDS (ATNC) DURING FERMENTATION

Formation of Apparent Total N-Nitroso Compounds (ATNC) was monitored throughout fermentations of all-malt ale worts supplemented with nitrate (0–100 mg Litre−1 (0–100 ppm)). The pitching yeasts were obtained from commercial breweries and contained different levels of the contaminant bacterium Obesumbacterium proteus (0–2.1% by number). Levels of ATNC present at the end of fermentation were dependent on both initial wort nitrate levels and the initial level of bacterial contamination of the pitching yeast. Only relatively low nitrate levels were required to produce ATNC levels greater than the Brewers' Society recommended limit of 20 μg Litre−1 (ppb), provided that the bacteria were present. Indeed, the use of whole hops alone would contribute sufficient nitrate to the wort to produce excessive amounts of ATNC, provided O.proteus was present. The only feasible solution to ATNC production during fermentation is to remove the contaminating bacteria from both the pitching yeast and brewing plant. Effective removal of O.proteus can be achieved by acid washing the pitching yeast under carefully controlled conditions, prior to fermentation.