Studies in gastric carcinogenesis. II. Absence of elevated concentrations of N-nitroso compounds in the gastric juice of Greek hypochlorhydric individuals.

The concentrations of nitrate, nitrite, N-nitroso compounds and bacteria were measured in 96 samples of fasting gastric juice, pH 0.90-8.50, obtained from 56 individuals just before or at various times (8 days - 1 year) after gastric operation. The mean pH of the post-operative samples [4.66 +/- 0.39 (SEM)] was significantly higher than that of the pre-operative ones [3.29 +/- 0.33 (SEM)]. A positive correlation with pH was observed for the concentrations of total and nitrate-reducing bacteria (median values 5.0 X 10(5) organisms/ml and 9.2 X 10(4) organisms/ml, respectively, for samples with pH greater than or equal to 1.2 X 10(3) organisms/ml and 0 organisms/ml, respectively, for samples with pH less than or equal to 2.5) and nitrite [mean values 22.5 +/- 3.1 (SEM) microM and 3.20 +/- 0.5 (SEM) microM for samples with pH greater than or equal to 6.5 and pH less than or equal to 2.5, respectively]. No correlation with pH was seen for the concentrations of nitrate [mean value 0.48 +/- 0.06 (SEM) mM] or N-nitroso compounds [mean value 0.30 +/- 0.06 (SEM) microM]. The concentrations of bacteria and nitrite, although increased in hypochlorhydric individuals, were lower than those reported for corresponding individuals in other, primarily British, studies. It is suggested that the relatively low concentrations of nitrite observed in our hypochlorhydric population may account for the absence of elevated concentrations of N-nitroso compounds and that the latter phenomenon may be related to the relatively low frequency of gastric cancer in Greece.

Studies in gastric carcinogenesis. III. The kinetics of nitrosation of gastric-juice components in vitro and their implications for the in vivo formation of N-nitroso compounds in normal and in hypochlorhydric populations.

Fasting human gastric juice was treated in vitro, at pH 2-7 and 37 degrees C for 2 h, with 5-100 microM sodium nitrite. Under these conditions (which simulated those occurring in vivo in normal or hypochlorhydric individuals), the formation of total N-nitroso compounds had the following characteristics: (i) it increased greatly at pH less than 3; (ii) it showed first-order dependence on nitrite concentration; (iii) it was faster at pH 7 than at pH 5. These observations are compatible with the N-nitroso compounds formed by the interaction of nitrite with gastric juice being N-nitrosamides or related compounds. Furthermore, based on the results of this study, it is suggested that in order for hypochlorhydria to give rise to increased formation of N-nitroso compounds in the stomach, it would be necessary for it to be accompanied by a greater than 5- to 10-fold increase in gastric nitrite concentration relative to that found in the normal population, a condition which is not necessarily fulfilled in all hypochlorhydric individuals or populations. The implications of this conclusion for the assessment of the role on gastric N-nitroso compounds in the etiology of gastric cancer are discussed.

Alternative bioactivation routes for beta-hydroxynitrosamines. Biochemical and chemical model studies.

The biochemical retroaldol-like fragmentation of beta-hydroxynitrosamines has been investigated further. The extent of fragmentation of 2-hydroxy-2-methylpropyl-methylnitrosamine (HMPMN) to N-nitrosodimethylamine and acetone induced by metabolic activation increases as the NADPH level is decreased. 2-Hydroxy-2-phenylethyl-methylnitrosamine (HPhEMN) undergoes competitive oxidation to 2-oxy-2-phenylethyl-methylnitrosamine (OPhEMN) and fragmentation to benzaldehyde and N-nitroso-dimethylamine in the presence of a metabolic activation system from rat liver. The extent of the oxidation was increased by preinduction of the rats with phenobarbital, or separate addition of NADPH and NAD, but was decreased by addition of dimethyl sulfoxide. The fragmentation was observed most readily when oxidation was inhibited or was not induced by cofactors. When HPhEMN was administered to a rat intraperitoneally, benzaldehyde (fragmentation) was found in the urine with OPhEMN and the substrate, but only the last two substances were found in liver and blood. These experiments provide evidence for retroaldol-like fragmentation of beta-hydroxynitrosamines both in vitro and in vivo. In a related investigation, it was found that N-nitroso-N-4-chlorophenyl-2-aminoethanal (NCAE) is extremely reactive and induces spontaneous generation of 4-chlorobenzenediazonium ion in chloroform, as trapped by 2-naphthol. NCAE reacts with dimethylamine in chloroform, benzene or methanol to give N-nitrosodimethylamine and 4-chloroaniline, among other products. This suggests that beta-nitrosaminoaldehydes produced by the biooxidation of their corresponding alcohols could produce cell alteration through alkylation, deamination or transnitrosation.

Nitrosamine formation from ternary nitrogen compounds.

When amides of pyrrolidine are heated with sodium nitrite, small amounts of NPYR are formed. Both the rate of reaction and the nitrosamine yield are increased when ethylene glycol and glycerine are used as solvents. Kinetic evidence suggests that these polyols react with the amide to form esters and the corresponding amine. The esters then react with sodium nitrite to give nitrite esters which rapidly nitrosate the amine. The heating of LDEA with sodium nitrite gives relatively high yields of NDELA in relatively short times and this nitrosation can be explained by this mechanistic hypothesis. 2-N,N-dimethylaminomethylpyrrole reacts very rapidly with nitrous acid at 25 degrees to give NDMA as the sole nitrosamine. This suggests a new mechanism of tertiary amine nitrosation.

New pathways for the rapid formation of N-nitrosamines under neutral and alkaline conditions.

Ethylene glycol, several carbohydrates (sugars) and alkanolamines influence the formation of carcinogenic N-nitrosamines in neutral and alkaline aqueous solutions at 25 degrees C in presence of dissolved nitrosyl gases. These compounds either catalyse or inhibit the reactions (depending on the experimental conditions and reagent reactivities) by forming a nitrite ester intermediate, which reacts readily with secondary amines. The reactions may explain the origin of some N-nitrosamines in vivo and in consumer products, particularly those originating from NOX pollutants. N-Nitrosamines are also formed at ambient temperatures by the gamma-radiolysis of neutral aqueous solutions of either NaNO2 or NaNO3 and secondary amines. With NaNO3, N-nitroamines are in accompanying product. These reactions are considered to proceed via N2O3 and N2O4 intermediates, generated from NaNO2 and NaNO3, respectively.

Rapid formation of N-nitrosamines from nitrogen oxides under neutral and alkaline conditions.

The formation of carcinogenic N-nitrosamines in neutral and alkaline aqueous solutions (pH 6-14) at 25 degrees C is reported using dissolved N2O3 and N2O4 gases. These reactions are very much faster than those with acidified nitrite: typically, 2 X 10(-3) M amine gives ca. 10-50% N-nitrosamine in a few seconds with 5-20 fold excess of nitrogen oxide. The N-nitrosamine yield in 0.1 M sodium hydroxide is independent of amine basicity from pKA 11.2-0.99, but decreases with decreasing pH of the reaction solution for the more basic amines. Significantly, N-nitrosamine yields are not lowered with diluted nitrogen oxides (1000 ppm) and moderately basic amines (eg. N-methylpiperazine) react readily at physiological pH. The mechanism by which these reactions occur is discussed, with particular reference to the existence of two reactive tautomeric forms of N2O3 and N2O4. The formation of carcinogenic N-nitrosamines from NO in ethanol at 25 degrees C is also reported. These reactions are slow in the absence of air (oxygen), I2 or metal salts. Oxygen accelerates nitrosation by converting NO via NO2 to either N2O3 or N2O4, but both I2 and metal salts are effective under anaerobic conditions, where reaction rates are virtually independent of amine basicity but depend on the nature of the added reagent. The most effective substance is I2, which gives quantitative yields of N-nitrosamine in a few minutes at 25 degrees C by forming the reactive nitrosyl iodide (NOI) reagent. Acceleration in ethanol at 25 degrees C is also observed with AgI, CuI, CuII, ZnII, FeIII and CoII salts, among others, with substantial amounts of N-nitrosamine being produced in ca. 30-300 min. Metal iodides intervene by way of the NOI reagent, as for I2, but other salts require a mechanism involving reaction between a metal-amine complex and NO, itself. The results show that carcinogenic N-nitrosamines may form under a much wider range of experimental conditions than suspected hitherto. Their relevance to human exposure is discussed, with particular reference to urban pollution and the effect of dietary antioxidants.