The formation of nitrite and N-nitroso compounds in salivas in vitro and in vivo.

The considerable differences in salivary nitrite levels observed previously in subjects who had abstained overnight from dietary nitrate as completely as possible were again found in tests on ten separate days. A 25-fold variation was observed in the rate of production of nitrite in vitro during the incubation of salivas from different subjects with 16 mM-nitrate under standardized conditions. N-Nitroso compounds were detected as a group on 11/100 and 14/100 occasions after incubations in vitro without and with added nitrate, respectively. No significant changes in salivary nitrite level were found 1 hr after the volunteers had ingested water-borne nitrate at approximately the World Health Organization recommended limit for continuous use (50 mg NO-3/litre), but increases in nitrite concentration were consistently found after volunteers ingested nitrate in water at four times this concentration. Although the nitrite concentrations were markedly increased following the intake of nitrate at the higher level, the occurrence of N-nitroso compounds in the salivas of the volunteers was greater immediately prior to the ingestion of water-borne nitrate than 1 hr afterwards.

Nitrate reduction, gastro-intestinal pH and N-nitrosation in gnotobiotic and conventional rats.

The reduction of nitrate in germ-free, gnotobiotic and conventional rats was investigated using blood methaemoglobin values as indicative of nitrite formation. Nitrate reduction was found to occur in the absence of a microbial flora, and throughout the experiment the blood content of methaemoglobin was higher in germ-free than in conventional rats. In vitro incubations of the gastric and small-intestinal mucosae of germ-free rats confirmed the presence of a heat-labile nitrate-reducing system. Measurement of the gastro-intestinal pH of germ-free and conventional rats revealed a generally higher pH value throughout the germ-free gastro-intestinal tract, with highly significant differences in the luminal pH of the forestomach, jejunum/ileum and caecum and a significant difference in the pH of the glandular stomach. Although some formation of N-nitrosoproline from proline and nitrate occurred in germ-free and gnotobiotic rats, nitrosation proceeded more readily in conventional rats. This effect may have been due to the lower gastric pH in the conventional rats although a more direct role for the flora cannot be discounted.

Characterization of new viridomycins and requirements for production in cultures of Streptomyces griseus.

Cultures of Streptomyces griseus grown under phosphate-limiting conditions produced a complex of green products. Three of these were separated from the mixture and characterized. One was identified as viridomycin A, the ferrous chelate of 4-hydroxy-3-nitrosobenzaldehyde; the second (actinoviridin A) was the corresponding carboxylic acid chelate and the third (viridomycin E) was a hybrid chelate containing both the aldehyde and acid ligands. Only two out of nine strains of S. griseus examined produced viridomycins and the ligands were biosynthesized only in media from which phosphate had been exhausted. Optimization of the production medium showed that fructose and alanine were the most favorable carbon and nitrogen sources and that relatively high concentrations of ferrous ions were necessary. The results suggest that viridomycins are not produced by S. griseus as iron scavengers in response to iron deficiency but as secondary metabolites that are stabilized adventitiously in the broth by metal ion chelation.

Transnitrosating activity of N-nitroso-N-methyl-p-toluenesulfonamide in rats and human gastric juice.

N-nitroso-N-methyl-p-toluenesulfonamide (NMTS, diazald, CAS 80-11-5) is a widely used compound for laboratory production of diazomethane. The present results showed that the noncarcinogenic NMTS reacts as a transnitrosating agent with amino nitrogen of secondary amines and amide both in vitro (human gastric juice) and in vivo (rats) to yield N-nitroso compounds. Since all compounds formed (NMOR, NDMA, NPIP, NPZ, NMU) are known animal carcinogens, caution should be taken by users handling NMTS.

[Carcinogenic hazard of small doses of nitrite in connection with the endogenous synthesis of nitroso compounds]. / Kantserogennaia opasnost' malykh doz nitrita v sviazi s éndogennym sintezom nitrozosoedinenii.

Carcinogenic risk of small doses of precursors of nitroso compounds, i.e. sodium nitrite (total dose in 0.2-2.0 g/mouse in drinking water) and morpholine (total dose is 0.23 g/mouse in bread) was studied in 520 CBA and 290 C57Bl mice during 96 weeks. It is shown that under these conditions the carcinogenic effect was more pronounced in CBA mice: there was a significant increase in general incidence of tumours, particularly liver tumours and hemoblastoses in CBA females and malignant liver tumours in CBA males.

[Factors influencing formation of dinitrosyl complexes of non-heme iron in the organs of animals in vivo]. / O faktorakh, vliiaiushchikh na obrazovanie dinitrozil'nykh kompleksov negemovogo zheleza v organakh zhivotnykh in vivo.

The formation of paramagnetic non-haem iron dinitrozyl complexes (2,03 complexes) in mouse liver has been detected, when these animals were exposed to a diet with high content of nitrite or nitrite and iron salt. These complexes appeared after 3-5 days and their content was kept at the same level during a month. The 2,03 complexes content in mouse liver was 3 times higher for mice which were exposed to the diet with nitrite and iron salt than for those on the diet only with nitrite. While the liver preparations of experimental mice were treated with NO in vitro the 2,03 complexes content increased in the liver and reached the same level for all experimental mice. It has been supposed that the 2.03 complexes formation was limited in mouse liver in vivo, both by NO production in the liver and by the content of the more labile form of non-haem iron, which was primarily incorporated into the complexes.

[Formation of nitrosyl complexes of nonheme iron (2.03 complexes) in animal tissues in vivo]. / Obrazovanie nitrozil'nykh kompleksov negemovogo zheleza (kompleksov 2.03) v tkaniakh zhivotnykh in vivo.

Formation or dinitrozyl non-haem iron complexes (2.03 complexes) in white rats' tissues in vivo has been studied. The formation of these complexes has been examined in the liver kidneys and small intestines when injecting p/o NaNO2 to the rats. Most of the complexes have been found in the liver. They have not been detected in the heart, spleen, muscle, cerebrum and marrow in vivo. 3-4-fold content of 2.03 complexes has been determined while introducing iron and NaNO2 in running water. This effect has not been found when introducing iron and NaNO2 simultaneously. The mechanism of 2.03 complex formation in the animal tissues in vivo has been proposed. According to this mechanism these complexes are formed in the tissues when Fe-NO from nitrozyl non-haem from complexes originating in blood passes into SH-group of protein in tissues. The formation of 2.03 complexes in vivo has been found in blood uniform elements.

Roles of cysteine as both precursor of thiazolidine 4-carboxylic acids found in human urine and possible inhibitor of endogenous N-nitrosation.

In order to compare the utility and significance of 2-R-N-nitrosothiazolidine 4-carboxylic acids excreted in human urine as an index for exposure to N-nitroso compounds, the differences in formation of N-nitrosothiazolidine 4-carboxylic acid (NTCA; R = H) and N-nitroso-2-methylthiazolidine 4-carboxylic acid (NMTCA; R = CH3) were studied in vitro. It was determined that NMTCA has a 3:1 trans:cis stereoisomer ratio, while NTCA has a 1:1 trans:cis ratio; nitrosation acts on a pH-dependent equilibrium mixture of cysteine and aldehyde in equilibrium with thiazolidine 4-carboxylic acid, with cysteine blocking N-nitrosation. Previous reports on 2-R-N-nitrosothiazolidine 4-carboxylic acids in human urine show widespread involvement of cysteine, which has a dual role with nitrosating species. In view of this and the rapid blocking of N-nitrosation and slow trans-nitrosation by cysteine at acid pH, it is suggested that there may be a hitherto unrecognized protective role of thiol functions in dietary constituents.

In vitro production of N-nitrosodimethylamine and other amines by proteus species.

N-nitrosodimethylamine, a potent carcinogen, was produced by three strains each of Proteus mirabilis, P. morganii, and P. rettgeri, but not by three strains of P. vulgaris grown under the same conditions. Many of the alkaline-extractable volatile metabolites elaborated by these organisms are the same, but there are some qualitative and quantitative differences among species. Representative gas-liquid chromatographic profiles of the four species are presented, and the significance of the differences is discussed. Primary emphasis, however, is given to the importance of the production of N-nitrosodimethylamine by these microorganisms and the conditions under which it is produced.

Nitrate and nitrite in normal gastric juice. Precursors of the endogenous N-nitroso compound synthesis.

The nitrate and nitrite levels of 75 gastric juice samples from young and healthy fasting volunteers were examined. For both parameters a dependence on the specific pH value of the secretion was detected. The rise of the nitrite level from normal 0.1 ppm in the acid to 1.4 ppm in the neutral range can be explained by the activity of the bacterial flora even in the healthy stomach, which has already been demonstrated earlier. The hitherto existing theory that nitrate originates exclusively from the salivary glands, and if not reduced to nitrite by bacteria in the mouth cavity, is brought into the stomach via swallowed saliva only, does not explain the observation that there is a rise in nitrate concentration in neutral juice, too. Further investigations have to be made to see whether there are other endogenous sources of nitrate except the salivary glands in the gastrointestinal tract. The environmental pollutant nitrate must be paid more attention to in future.

Formation of carcinogenic N-nitroso compounds in corn-bread inoculated with fungi.

DMNA, DENA and MBNA are formed in corn-bread which has been inoculated with the most common species of fungi found in foodstuffs of Lin Xian County, Henan Province, such as F. moniliforme, A. flavus and others, and then, added with small amount of NaNO2 after a few days of incubation. These carcinogenic N-nitroso compounds can induce cancer of the liver or/and esophagus in experimental animals. Results of the present study show the capability of some fungi to produce chemical carcinogens as well as mycotoxins in contaminated food, and thus, open a new field of research in cancer etiology.

Endogenous formation of N-nitroso compounds: a current perspective.

Exposure of people to nitrosating agents occurs through multiple pathways, ranging from nitrogen dioxide reactions in the lung to acid-catalysed nitrosation in the stomach to nitrosation mediated by macrophages or bacteria. The use of N-nitrosoproline (NPRO) as an index of endogenous nitrosation has proved to be especially valuable for some of these pathways but may not be universally indicative. Since the development of the NPRO test by Ohshima and Bartsch in 1981, several forms of the test have been used for different purposes. This paper examines some of the issues related to endogenous nitrosation and NPRO and attempts to view the immediate future.

Model risk analysis of nitrosatable compounds in the diet as precursors of potential endogenous carcinogens.

The potential health risk posed by the endogenous formation of N-nitroso compounds (NOC) from nitrosation of dietary ureas, guanidines, amides, amino acids and amines (primary, secondary and aromatic) was estimated according to the model: Risk = [daily intake of precursor] X [gastric concentration of nitrite]n X [nitrosatability rate constant] X [carcinogenicity of derivative]. The daily intakes of these compound classes span five orders of magnitude (100 g/day amides, top; 1-10 mg/day secondary amines, ureas, bottom); the nitrosation rate constants span seven orders of magnitude (aryl amines, ureas, top; amides, secondary amines, bottom); and the carcinogenicity estimates span a 10,000-fold range from 'very strong' to 'virtually noncarcinogenic'. The resulting risk estimates likewise span an enormous range (nine orders of magnitude): dietary ureas and aromatic amines combined with high nitrite concentration could pose as great a risk as the intake of preformed N-nitrosodimethylamine in the diet. In contrast, the risk posed by the in-vivo nitrosation of primary and secondary amines is probably negligible. The risk contributed by amides (including protein), guanidines and primary amino acids is intermediate between these two extremes.

[Effect of nontoxic inhibitors on the nitrosation of drugs under conditions simulating the human stomach]. / Wirkungen einiger nicht-toxischer Inhibitoren auf die Nitrosierung von Arzneimitteln unter simulierten Bedingungen des menschlichen Magens.

The influence of sulphamic acid, sulfanilamide, p-aminosalicylic acid and ascorbic acid on the nitrosation behaviour of Analgin (active agent: noramidopyrinmethane sulfonate) was investigated in the pH range between 1.2 and 6.0 under simulated conditions of the human stomach. Using a colorimetric measuring method, the three last-mentioned compounds proved suitable as nitrosation inhibitors; preference should be given to ascorbic acid because of its better inhibitory capacity in the very weakly acidic pH range. Sulphamic acid is inadequate because it stimulates nitrosation in this pH range. The distinct inhibitory action of ascorbic acid was verified under simulated conditions of the human stomach in further 9 drugs undergoing nitrosation at different rates, which contain the active agents: aminophenazone, clomipramine, imipramine, desipramine, ampicilline, oxacilline, phenoxymethylpenicilline, ethambutole or piperazine. Despite the use of high doses of ascorbic acid, no complete inhibition of nitrosation could be achieved in the majority of drugs investigated.