Influence of dietary protein and gut microflora on endogenous synthesis of nitrate and N-nitrosamines in the rat.

Groups of four germ-free (GF) and conventional (CV) rats were given purified diets containing either 50 or 200 g lactalbumin/kg for 2 wk and their urinary excretion of nitrate was measured. Urinary excretion of N-nitrosoproline was also measured in one of the three experiments. Both GF and CV rats given the high-protein diet excreted significantly more nitrate and N-nitrosoproline than those given the low-protein diet. On both diets GF rats excreted more nitrate than their CV counterparts but N-nitrosoproline excretion was not affected by environment. Groups of 11 GF and CV rats given diets containing sesame meal with or without a supplement of lysine-HCl for 2 wk, excreted similar amounts of nitrate on both diets, but more nitrate was excreted by GF rats than by their CV counterparts. N-nitrosoproline excretion by rats given the lysine supplement was higher in both environments. It is concluded that endogenous synthesis of nitrate is mediated by mammalian tissues rather than microflora and that dietary protein is an important source of nitrogen for the synthesis, although surplus amino acids from an imbalanced protein source do not act as precursors of endogenously formed nitrate. Some of the synthesized nitrate or its precursors appears to be metabolized by the microflora in the CV rat.

Initiation activity of endogenously synthesized N-nitrosobis(2-hydroxypropyl)amine in the rat liver.

The initiation potential of N-nitrosobis(2-hydroxypropyl)amine (NDHPA) endogenously synthesized from bis(2-hydroxypropyl)amine (DHPA) or tris(2-hydroxypropyl)amine (THPA) in the presence of sodium nitrite (NaNO2) was investigated in the rat liver by quantitation of hepatocellular foci showing phenotypic expression of glutathione S-transferase placental form (GST-P). The investigation consisted of two experiments. In the first, male Wistar rats were divided into six groups as follows: group 1 was non-treated; groups 2 and 3 received 0.15% and 0.3% NaNO2, respectively; group 4 received 1% DHPA; groups 5 and 6 received 1% DHPA together with 0.15% and 0.3% NaNO2, respectively. In experiment 2, the same protocol was used except that 2% THPA was substituted for 1% DHPA. The treatments were continuous until sacrifice at week 94 in experiment 1 and week 104 in experiment 2. As a result putative preneoplastic GST-P-positive foci observed in the liver and increased dose-dependently in rats from groups 5 and 6 which had received DHPA and NaNO2, but not in rats administered THPA and NaNO2. The results indicate that endogenously synthesized NDHPA from DHPA and NaNO2 is capable of initiating neoplastic development in the rat liver.

Nitrite-cured meats as a source of endogenously and exogenously formed N-nitrosoproline in the ferret.

Nitrite-cured meat containing 120 mg Na15NO2/kg was fed to male ferrets (Mustela putorius furo). During consumption of the meat, the animals were dosed orally with 0.87 mmol [2-2H]proline. All urine was collected throughout the study and analysed for total N-nitrosoproline (NPRO) and isotopic enrichment of NPRO by mass spectrometry. The cured-meat diet increased the urinary excretion of NPRO 14-fold. Isotope analyses indicated that approximately 70% of the NPRO came from the cured meat, the majority of which was analytically unavailable or 'bound' NPRO in the meat. A small portion of the excreted NPRO appeared to be formed in the stomach as a result of ingesting the cured meat. A minor amount of the excreted NPRO did not contain any isotopically labelled atoms. The administration of ascorbic acid did not significantly alter NPRO excretion. Animals dosed orally with 11.4 mumol of a peptide in which the N-terminal proline was nitrosated increased their excretion of NPRO by 385 nmol over the following 48 hr. These data indicate that nitrite-cured meat contains bound NPRO which contributes to the total amount of NPRO in the urine.

[Assessment of the degree of carcinogenicity of small doses of nitrites]. / Otsenka stepeni kantserogennoi opasnosti malykh doz nitritov.

Chronic experiments on CBA and C57B1 mice and acute experiments on CBA mice established: (a) carcinogenic effect of sodium nitrite given continuously with drinking water (0.1; 1.0 and 10.0 maximum allowable concentration) in combination with morpholine fed with bread, and (b) endogenous synthesis of nitrosomorpholine as a result of simultaneous intragastric administration of same doses of sodium nitrite and morpholine. Also, nitrosomorpholine and N-nitrosodimethylamine synthesis was observed in vitro following addition of low-dose sodium nitrite, morpholine and amidopyrine to human gastric juice. Carcinogenic hazard associated with low-dose nitrite consumption in humans is discussed.

Lack of detectable N-nitroso-N-methyl-N-cyclohexylamine in humans after administration of bromhexine.

The possible formation of N-nitroso-N-methyl-N-cyclohexylamine (NMCA) from the drug bromhexine (N-methyl-N-cyclohexyl-(2-amino-3,5-dibromobenzyl)-ammonium hydrochloride) and nitrite was investigated in humans using three different approaches: 1. analysis on metabolites of NMCA in human urine; 2. analysis on NMCA in human gastric juice; 3. in vitro incubation of human gastric juice with therapeutic bromhexine doses. Diet given to volunteers was varied during these investigations with respect to nitrate content. Experiments with a maximum load of 200 mg nitrate to stimulate nitrite formation were performed. Results of in vivo experiments did not indicate any formation of NMCA. In one out of 39 ex-vivo/in-vitro experiments (with a load of 100 mg nitrate in drinking water) 0.5 ng NMCA/ml gastric juice could be detected which is near the detection limit. Finally, this study showed that bromhexine is not secreted by saliva. This allows to conclude that nitrite and bromhexine do not reach the stomach simultaneously over a longer period of time. In consequence, medication with bromhexine is not regarded to represent a risk due to nitrosamine formation.

Nitrosation of bromhexine and aminophenazone in gastric juice after high nitrate loading in the diet. An ex-vivo/in-vitro study in humans.

In an ex-vivo study, nitrosation of bromhexine (N-methyl-N-cyclohexyl-(2-amino-3,5-dibromobenzyl)-ammonium hydrochloride) in human gastric fluid under conditions of high dietary nitrate intake was investigated. 26 healthy volunteers received 200 mg of nitrate in a vegetable during a standard breakfast. Nitrite values in saliva were significantly increased 2 to 3 h after nitrate intake. In contrast, nitrite levels in gastric fluid sampled 3 h after nitrate intake remained in the low concentration range of less than or equal to 1.7 micrograms/No2-/ml. Incubation of gastric fluid samples at 37 degrees C with the recommended maximum single oral dose of bromhexine (16 mg/100 ml) or an equimolar concentration of aminophenazone revealed in 4/26 cases formation of N-nitrosomethylcyclohexylamine from bromhexine at barely detectable levels. In contrast, aminophenazone generated N-nitrosodimethylamine at a very high rate, resulting in yields of greater than 50% in several samples. In view of the average daily background exposure to preformed nitrosamines in foods (0.5-1 microgram/capita), according to these results the possible contribution of in-vivo nitrosation of bromhexine corresponds at best to 10% in addition to the daily background exposure and therefore can be regarded as negligible.

Kinetic and analytic investigations on the formation of N-nitroso-N-methyl-N-cyclohexylamine from bromhexine and nitrite.

Bromhexine (N-methyl-N-cyclohexyl-(2-amino-3,5-dibromobenzyl)-ammoniumhydr ochloride) forms N-nitroso-N-methyl-N-cyclohexylamine (NMCA) under the conditions of the WHO Nitrosation Assay Procedure (NAP-test). The formation kinetics of this compound was investigated. The formation of NMCA depends on the square of the nitrite concentration. The reaction has a narrow pH-optimum at pH 3. The reaction is quick: After 1 h about 70% of the maximum amount of NMCA is formed. To study this reaction kinetics sensitive assays with a detection limit up to 0.5 ng/ml NMCA were developed. The stability of the components of the system, especially that of NMCA and nitrite, were further studied. The latter is rather instable under conditions found in an acidic stomach.

Nitrosamine formation by denitrifying and non-denitrifying bacteria: implication of nitrite reductase and nitrate reductase in nitrosation catalysis.

Biochemical, microbiological and genetic studies were done to characterize the mechanism of bacterial formation of N-nitrosomorpholine (NMOR) from morpholine and nitrite at neutral pH. In Escherichia coli and Proteus morganii, the nitrosating activity was markedly induced when bacteria were cultured under anaerobiosis in minimal medium containing nitrate, while in the presence of nitrite there was no induction. However, induction of the nitrosating activity in Pseudomonas aeruginosa occurred in anaerobic cultures in the presence of either nitrate or nitrite. The nitrosation capacity was also examined in various E. coli K12 mutants whose structural gene of either nitrate reductase or nitrite reductase was deleted. Nitrosation was not linked to the three (NADH-, formate- and glucose-dependent) nitrite reductases but was directly dependent on the presence of a nitrate reductase.

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.

Nitrate as a precursor of the in-vivo formation of N-nitrosomorpholine in the stomach of guinea-pigs.

Reduction of nitrates to nitrites and formation of the carcinogen N-nitrosomorpholine (NMOR) was investigated in the stomach of guinea-pigs. A semisynthetic diet with nitrate plus morpholine was administered intragastrically after a 24-h fast; after treatment, the animals were killed and stomach nitrite contents were determined 6, 12, 18, 24 and 30 min after the treatment using a colorimetric method. NMOR content was determined 18 min after treatment with nitrate plus morpholine using gas chromatography-thermal energy analysis. Reduction of nitrates to nitrites in the stomach was observed that was sufficient to synthesize NMOR in guinea-pigs under the conditions of this experiment.

[Induction of papilloma and carcinoma in the forestomach of mice by in vivo formation of N-3-methylbutyl-N-1-methylacetonylnitrosamine (MAMBNA)].

Forestomach papilloma and carcinoma, as well as liver lesion, were induced in mice by gavaging precursors of the new nitrosamine, MAMBNA (N-3-methylbutyl-N-1-methylacetonylnitrosamine) and NaNO2. The results were-similar to those in mice and rats fed on preformed MAMBNA compound. However, the induction of such tumors by in vivo formation of MAMBNA required longer time and much larger doses. Moreover, the lesions of epithelial hyperplasia in the urinary bladder, lymphoid tumor, intestinal carcinoma and interstitial-cell tumor of the testis also developed in some of the experimental animals. This may indicate that the intragastric synthesis of MAMBNA is less effective in the production of forestomach tumors in mice.

Identification of cholesterol as a mouse skin lipid that reacts with nitrogen dioxide to yield a nitrosating agent, and of cholesteryl nitrite as the nitrosating agent produced in a chemical system from cholesterol.

The skin lipids of mice exposed to nitrogen dioxide (NO2) and mouse skin lipids exposed in vitro to NO2 contain nitrosating agents (NSAs), that react with amines to produce nitrosamines. This situation represents a potential hazard of exposure to NO2. A principal NSA precursor in mouse skin lipids was purified by thin-layer and high-performance liquid chromatography. Each fraction was assayed by bubbling in NO2 and determining NSA. The precursor was identified as cholesterol on the basis of its chromatographic behavior and spectral properties. In a chemical system, cholesterol reacted with NO2 to give 13% yields of an NSA, which was identified from its spectral properties as the previously known compound, cholesteryl-3-beta-nitrite. These findings and the chromatographic behavior of a major NSA in the skin lipids of NO2-exposed mice suggested that this NSA was cholesteryl nitrite.

Inhibitory effect of alpha-tocopherol on the formation of nitrosomorpholine in mice treated with morpholine and exposed to nitrogen dioxide.

The possibility that alpha-tocopherol (vitamin E) inhibits the formation of nitrosomorpholine (NMOR) in vivo was investigated in mice orally pretreated with alpha-tocopherol (2.5-100 mg/kg body wt) once daily for 6 days. Twenty-four hours later, the animals were injected i.p. with 2 mg of morpholine (MOR) per animal followed by exposure to 47 p.p.m. of NO2 for 2 h. Under these conditions, low oral doses of alpha-tocopherol (2.5-5 mg/kg body wt) significantly decreased NMOR formation in vivo. As total body alpha-tocopherol levels increased, in vivo NMOR formation decreased, and a maximal 50-70% inhibition of NMOR formation occurred at alpha-tocopherol levels of 5 micrograms/g body wt. Additional results showed that NMOR was rapidly eliminated in mice, so that studies which measure the levels of NMOR found in animals treated with MOR and then exposed to NO2 may underestimate the amount of NMOR that is actually formed. Finally, oral pretreatment of up to 100 mg of alpha-tocopherol/kg body wt had no effect on NMOR elimination.

Synthesis of nitrosomethylisoamylamine from isoamylamine and sodium nitrite by fungi.

Nitrosomethylisoamylamine (NMIA), a carcinogenic N-nitroso compound was synthesized from isoamylamine (IAA) in a glucose-ammonium nitrate medium after several days' incubation with fungi and subsequent nitrosation with sodium nitrate. The nitrosamine was not produced by control reactions which lacked either IAA or fungi. The yield of NMIA varied with the length of incubation after inoculation with the fungi, and with the concentrations of IAA and NaNO2, the duration of nitrosation, the pH of the medium and the species of the fungi. The optimum conditions for the formation of the nitrosamine are reported.

Formation of bacterial mutagens from the reaction of chewing tobacco with nitrite.

Using the Salmonella/microsome assay system, the mutagenicity of chewing tobacco extracts (CTE) treated with and without sodium nitrite under acidic conditions was examined. Mutagenic activity was found only for nitrite-treated CTE in both tester strains, TA98 and TA100, and was independent of metabolic activation. Formation of mutagenic substances from CTE by nitrite was dependent on acidic pHs (the highest at pH 2) and could be inhibited by ascorbate. The mutagenic potency of CTE plus nitrite was proportional to the content of nitroso compounds generated in the reaction mixture, indicating that the nitrosation process was involved. The possible in vivo nitrosation and the potential health effect are discussed.

Effect of vitamins C and E on endogenous synthesis of N-nitrosamino acids in humans: precursor-product studies with [15N]nitrate.

The endogenous formation of nitrosoproline (NPRO) following administration of nitrate and proline is reported in ten healthy young adults. There was a relatively constant basal excretion of NPRO, 26 +/- 10 (SD) nmol/day, in excess of amounts found in the diet. This basal synthesis of NPRO was not reduced by ascorbic acid (2 g/day) or alpha-tocopherol (400 mg/day). A significant rise in the excretion of NPRO was observed following the administration of nitrate and proline, ranging from 29 to 318 nmol/24 h with a mean of 100 nmol/24 h. [15N]Nitrate was used as a tracer to study the observed excess excretion of NPRO in urine. The data revealed that urinary NPRO excretion as a result of endogenous synthesis is not totally derived from ingested nitrate as its precursor. The ingestion of ascorbic acid and alpha-tocopherol inhibited the incorporation of [15N]nitrate into NPRO by 81 and 59%, respectively. An additional nitrosamino acid, N-nitrosothiazolidine-4-carboxylic acid, was present in the urine. It was found that N-nitrosothiazolidine-4-carboxylic acid increased 6-fold upon ingestion of nitrate. Ascorbic acid and alpha-tocopherol blocked this nitrate induced synthesis.