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.     

Alkylating potency of nitrosated amino acids and peptides.

The alkylating potency of unstable N-nitrosamino acids and N-nitrosopeptides was investigated in vitro using 4-(para-nitrobenzyl)pyridine (NBP) as nucleophile. Of the amino acids, Met and those with an aromatic side chain were the most potent. The relative overall alkylating potency was 23:10:5:4:2:1: for Trp, Met, His, Tyr, Phe and Gly, respectively. The homo-dipeptides were much more potent than the amino acids, with relative potencies of 400:110:100:8:3:1, for Trp-Trp, Tyr-Tyr, Met-Met, Asp-Asp, Phe-Phe and Gly, respectively. In the one-phase reaction system (in which NBP is already present during the nitrosation reaction at acidic pH), all amino acids tested showed a second-order reaction for nitrite. In the two-phase system (in which NBP is added only after bringing the nitrosation reaction mixture to neutrality), all amino acids tested except one again showed a second-order reaction for nitrite (Phe, His, Asp and the dipeptide artificial sweetener aspartame); only Met under these conditions had a reaction order of one for nitrite. This could mean that nitrosation of the side chain of Met produces a second N-nitroso product which is relatively stable in acid but reacts with NBP under neutral conditions. In the human stomach, this side-chain nitrosation might become more important than the reactions at the primary amino group, firstly because of the greater stability of the product(s) in acid and secondly because of the first-order reaction rate for nitrite.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nitrosation of dietary precursors

The diet contains a large number of constituents which can be nitrosated in the gastrointestinal tract (especially in the stomach) to potentially carcinogenic nitroso compounds (NOC). The nitrosation of food mixtures has been investigated with a number of assays, such as chemical analysis or detection of alkylating potential, mutagenicity and carcinogenicity. Relatively good information is available on the formation of stable nitrosamines using high nitrite concentrations. Little is known, however, about the formation of chemically unstable NOC at low nitrite concentration and their genotoxicity in target cells. A comparison of the precursor classes, alkylamines, aromatic amines, amino acids, amides and peptides, ureas and guanidines, reveals a vast range, both with respect to daily intake (10(5)-fold) and nitrosation rate (10(4)-fold both for 1st and 2nd order nitrite dependence). A total span of 10(8) results for the relative yield of NOC in the stomach. The endogenous NOC burden from dietary ureas and aromatic amines may represent as large a hazard as the intake of preformed NOC. Recent evidence also indicates that heterocyclic amines and phenols must be considered and that the half-life of nitrosated alpha-amino acids can be much longer than that of nitrosated primary alkylamines. In these classes, more information should be collected on dietary concentrations, on the nitrosation under realistic conditions and on the genotoxicity in stomach lining cells. Within a chemical precursor class, a wide range is seen with respect to alkylating potency. It cannot, therefore, be excluded that individual precursors within the top ranking classes might become more important than single preformed NOC. Not considered in the above analysis but probably just as important for a risk evaluation in a population is the knowledge of the nitrosation conditions and target cell susceptibility in individuals.     

Possible underestimation of nitrosatable amine levels in artificial saliva extracts of children's rubber pacifiers and baby-bottle teats

Children's pacifiers and baby-bottle nipples from various countries were analysed for their content of N-nitrosamines and nitrosatable amines. Using a method involving extraction with artificial saliva, several nitrosamines including N-nitrosodi-n-butylamine (NDBA), N-nitrosodiethylamine (NDEA), N-nitrosodimethylamine (NDMA) and N-nitrosomorpholine (NMOR) were detected in addition to the three nitrosatable amines dibutylamine (DBA), diethylamine (DEA) and dimethylamine (DMA). Upon nitrosation in artificial saliva, these amines produced not only the related N-nitrosamines but also relatively high levels of the corresponding nitramines--N-nitrodibutylamine (NTDBA), N-nitrodiethylamine (NTDEA) and N-nitrodimethylamine (NTDMA). Thus, both N-nitramines and N-nitrosamines should be measured after nitrosation; otherwise, the method probably underestimates the quantities of nitrosatable amines present in artificial saliva extracts. Whether N-nitramines, some of which have been shown to be both mutagenic and carcinogenic, are formed in the saliva of babies exposed to these products remains to be confirmed.     

Inhibitory effects of phenolics,teas and saliva on the formation of mutagenic nitrosation products of salted fish

The objectives of this study were to simulate in vitro some of the conditions that may prevail in man during the ingestion of a meal and to quantitate the inhibitory effect of phenolics and phenolic-containing beverages on the formation of mutagenic nitrosation products. The test system consisted of nitrosating (pH 2, 1 h, 37°C) an aqueous fraction of a salt-preserved Chinese fish (Pak Wik) with or without the inhibitors to be tested and estimating the frequency of his⁺ revertants per survivor of Salmonella typhimurium (strain TA1535). The phenolics and teas were added to the nitrosation mixture. Catechin, chlorogenic acid, gallic acid, pyrogallol and tannic acid suppressed the formation of mutagenic nitrosation products. The inhibitory efficiency was comparable to that of ascorbic acid. A Japanese, a Chinese and a Ceylonese tea also prevented the formation of mutagenic nitrosated fish products at doses which are usually consumed by man. Moreover, saliva exerted an inhibitory effect. The inhibitory effect was not additive when the phenolics or saliva were added concurrently to the nitrosation mixture. The possibility that phenolics are involved in the apparent chemopreventive effect of fruits and vegetables is discussed.     

Nitrosation by stimulated macrophages. Inhibitors, enhancers and substrates

Macrophages and their immortalized cell lines can be activatedto form nitrite and nitrate via oxidation of arginine and thisis accompanied by the formation of N-nitroso compounds. Themechanism of nitrosamine formation has been investigated throughthe use of compounds which are known either to inhibit or enhanceacid-catalyzed nitrosation. The range of nitrogen acceptorshas been expanded to include ureas as well as amines of varyingpK_a and structure. The results are consistent with a mechanismin which NO is oxidized to N₂O₃ and N₂O₄, which are capableof nitrosating amines, but not ureas or amides, at neutral pH.This is in agreement with a recent observation that macrophagecell-free extracts can oxidize arginine to NO. The effect ofascorbic acid on intact activated macrophages is complex sincenitrite formation is enhanced over a very wide range of ascorbateconcentrations (5–500 µM) while nitrosation is inhibitedat ascorbate concentrations >50 µM.     

Amadori- and N-nitroso-Amadori compounds and their pyrolysis products. Chemical, analytical and biological aspects

N-(1-Deoxy-D-fructos-1-yl)-L-amino acids (fructose amino acids), Amadori compounds, are formed by reaction of D-glucose and L-amino acids and Amadori rearrangement. They are detected in heat-processed natural products and various foodstuffs and are key products of the Maillard-Browning reaction. N-Nitroso-N-(1-deoxy-D-fructos-1-yl)-L-amino acids (N-NO-fructose amino acids), N-NO-Amadori compounds, are formed in high yields by reacting fructose amino acids with sodium nitrite in acidic aqueous solution. They constitute a new class of non-volatile, bis-beta-oxidized nitrosamine derivatives with unknown biological (mutagenic and/or carcinogenic) activity. N-NO-Fructose amino acids may be formed in nitrite-containing Maillard systems (e.g., cured-meat products, tobacco) or in the human stomach after ingestion of food containing fructose amino acids and nitrite in food or saliva. Thirteen fructose amino acids and 13 N-NO-fructose amino acids (-gly, -ala, -val, -leu, -ileu, -ser, -thr, -met, -asp, -pheala, -tyr, -his, -trp) were prepared and investigated by high-resolution 1H-nuclear magnetic resonance (NMR) and 13C-NMR spectroscopy. The percentage amounts of the sugar ring forms (beta-pyranose, beta-furanose, alpha-furanose and alpha-pyranose) of these compounds in D2O mutarotation equilibrium were determined by 13C-NMR spectroscopy, together with the amounts (%) of E/Z isomers in the case of N-NO-compounds. The nitrosation products of D-fru-L-tyr, D-fru-L-his and D-fru-L-trp were isolated and identified by spectroscopic methods (NMR, infra-red). The N-NO-fructose amino acids can be separated by reversed-phase, ion-pairing, high-performance liquid chromatography. In some case the E/Z isomers are separated.     

Dietary fibre, fermentation and large bowel cancer

Diet, especially the amount of starch and dietary fibre which escape digestion in the small intestine, are major determinants of colon function in man. These carbohydrates are the principal substrates for fermentation by the large bowel flora. Carbohydrate fermentation results in lowered caecal pH and the production of short chain fatty acids of which butyric acid may protect the colon epithelium from dysplastic change. Protein digestion and amino acid fermentation also occur in the large bowel but the nature of its endproducts varies in relation to the amount of carbohydrate available. During active carbohydrate breakdown amino acid fermentation endproducts such as ammonia are used by the bacteria for protein synthesis during microbial growth, but in carbon-limited fermentation amines, ammonia, phenols and indoles, etc, accumulate. Fermentation also results in changes in colon pH which alters the metabolism of bile acids, nitrate, sulphate and other substances. Fermentation is thus controlled to a great extent by substrate availability, especially of carbohydrates which are derived from the diet. The potential to induce mutagenic change in colon epithelial cells and promote tumour growth may readily be influenced by diet.     

Nitrosation of tertiary aromatic amines related to sunscreen ingredients.

Possible routes to the formation of the sunscreen contaminant, 2-ethylhexyl 4-N-methyl-N-nitrosoaminobenzoate, have been investigated in a study of the nitrosation chemistry of 2-ethylhexyl 4-N,N-dimethylaminobenzoate (Padimate-O) and related tertiary and secondary amines. Padimate-O and the corresponding ethyl ester nitrosate rapidly at 25 degrees C in either N2O3:ether or HNO2:HOAc to produce a mixture of alkyl 4-N-methyl-N-nitrosoaminobenzoate and alkyl 4-N,N-dimethylamino-3-nitrobenzoate, the former of which is the major product. The nitrosative dealkylation of these amines at this low temperature is unusual. Asymmetrical amines exhibit a preference for nitrosative demethylation (methyl versus ethyl or benzyl), but the cleavage ratios in N2O3:ether are time-dependent, suggesting competing mechanisms with different reactant kinetic orders. A radical cation route would explain the unusual reactivity, which may compete with the established nitrosative dealkylation mechanism. 2-Ethylhexyl 4-N-methyl-N-nitrosoaminobenzoate was mutagenic in two strains of Salmonella typhimurium in the Ames assay.     

Formation of N-nitrosoiminodialkanoic acids and their unsuitability as biological monitors for endogenous nitrosation of dipeptides

Nitrosation of dipeptides that do not contain imino amino acids leads to rearrangement and formation of N-nitrosoiminodialkanoic acids. The optimal pH is 2.0 (0.8-3.2% yield). Under normal gastric conditions, a maximum yield of 0.1 mumol total N-nitrosoiminodialkanoic acid would be obtained for a typical dietary intake of 0.1 mol dipeptide. This corresponds to a total concentration of about 20 micrograms N-nitrosoiminodialkanoic acid/l gastric juice over a 24-h period. N-Nitrosoiminodialkanoic acids are excreted quantitatively in urine when fed by gavage to rats; however, they were not detected in normal human urine. It was concluded that determination of these compounds in human urine is not a suitable method for monitoring endogenous nitrosation of dipeptides.     

Accelerating effect of ascorbic acid on N-nitrosamine formation and nitrosation by oxyhyponitrite.

The reaction of nitrite ion with ascorbic acid and its effect on the rate of nitrosation of secondary amines have been investigated by differential pulse polarography in aqueous acidic solution. Ascorbic acid shows nonuniform behavior: it accelerates the nitrosation of N-methylaniline between pH 1.00 and 1.95, allows the nitrosation of diphenylamine and iminodiacetonitrile, but inhibits the nitrosation of secondary amines, such as dimethylamine, diethylamine, proline, hydroxyproline, N-methylaminoacetonitrile, N-methylaminopropionitrile, and sarcosine. The nitrosating agent generated by the reaction between ascorbic acid and nitrite ion appears to be oxyhyponitrite ion (N2O3-2).     

Catalysis and inhibition of N-nitrosation reactions

A number of factors that can lead to an acceleration or inhibition of N-nitrosation reactions may play an important role in the formation of N-nitroso compounds in foods and other environmental samples and in the body. The anion thiocyanate is a particularly effective catalyst for nitrosamine formation. It occurs in normal human saliva, but at higher concentrations in the saliva of smokers. Although nitrosation reactions are accelerated in the normal manner with increasing temperature, rate enhancements are also observed in frozen systems, a phenomenon that may be important in frozen foods. Surfactants that form micellar aggregates can accelerate the nitrosation of hydrophobic amines. This may be important in foods in view of the presence of components such as lecithin, or in the body in view of the occurrence of bile acid micelles. Nitrosation reactions may also be accelerated in the presence of certain carbonyl compounds, thiols and nitrosophenols. A number of compounds readily convert nitrosating agents into innocuous products and hence inhibit nitrosation reactions. If reductones such as ascorbic acid are present, they may react much faster with nitrous acid than amines and therefore effectively scavenge the nitrosating agent. Rapid reaction of polyhydroxyphenols with nitrous acid is another example of how endogenous or exogenous nitrosation reactions may be inhibited. Many microorganisms can influence nitrosation reactions by converting nitrate to nitrite, a reaction that can take place in foods, but which is particularly important in the body.     

N-nitroso compound formation in human gastric juice

The gastric formation of N-nitroso compounds probably constitutes a major source of human exposure to this important class of environmental carcinogens. Following reduction of nitrate to nitrite by oral or gastric bacteria, reaction with nitrogenous constituents of gastric juice can occur leading to the in situ formation of N-nitroso compounds, probably primarily derived from amides, ureas or aromatic amines. While gastric nitrite concentrations are raised in the achlorhydric relative to the normal stomach, the latter, owing to its acidity, offers a particularly favourable environment for the formation of N-nitroso compounds, as indicated by the finding of greatly increased gastric concentrations of N-nitroso compounds following an oral dose of nitrate. This illustrates the importance of the dynamic nature of the relationships between the various parameters involved in the formation of N-nitroso compounds. While in principle the same is true of the process of inhibition of nitrosation by reducing agents such as ascorbic acid (since, depending on the relative concentrations of reducing agent, nitrite and oxygen, inhibition or catalysis of nitrosation can occur), ingestion of 1 g ascorbic acid brings about a significant reduction in the gastric concentration of N-nitroso compounds.     

Involvement of free radicals in the formation of heterocyclic amines and prevention by antioxidants.

It was found that free radical intermediates, pyrazine cation radial and carbon-centered radicals, generated in the Maillard reaction of sugars/amino acids, were involved in the production of mutagenic and carcinogenic imidazoquinoquizaline heterocyclic amines. Prevention of the generation of these radical intermediates by phenolic antioxidants effectively inhibited the generation of the heterocyclic amines.     

Nutrition and dietary carcinogens

Three major factors for human carcinogenesis are (i) cigarette smoking, (ii) infection and inflammation and (iii) nutrition and dietary factors. Nutrition and dietary factors include two categories, namely genotoxic agents and constituents including tumor promotion-associated phenomena. This article first describes the genotoxic agents as microcomponents. These are mutagens/carcinogens in cooked food, fungal products, plant and mushroom substance, and nitrite-related materials, polycyclic aromatic hydrocarbons and oxidative agents. Emphasis has been given to heterocyclic amines (HCAs) to which humans are continuously exposed in an ordinary lifestyle. HCAs in food are mainly produced from creatin(in)e, sugar and from amino acids in meat (upon heating). They are imidazoquinoline and imidazoquinoxaline derivatives and phenylimidazopyridine. HCAs are pluripotent in producing cancers in various organs including breast, colon and prostate. Discussion is also given to plant flavonoids which are mutagenic but not carcinogenic. As a macrocomponent, overintake of total calories, fat and sodium chloride is discussed from the viewpoint of the increase of genetic alterations in tissues and of tumor promotion-associated issues. Studies of nutrition and dietary condition will eventually lead us to cancer prevention, namely delay of onset of cancer to the late phase of human life, which is called 'natural-end cancer' (Tenju-gann).     

Activation of some aromatic amines to mutagenic products by prostaglandin endoperoxide synthetase

Cooxidation of xenobiotics may occur during prostaglandin biosynthesis. The ability of prostaglandin endoperoxide synthetase to cooxidize several aromatic amines and other chemicals to mutagenic products was tested with the standard Salmonella tester strains. The microsomal fraction of ram seminal vesicles, a rich source of prostaglandin endoperoxide synthetase, in the presence of the prostaglandin endoperoxide synthetase substrate arachidonic acid metabolized benzidine, 2-aminofluorene, 2-naphthylamine, and 2,5-diaminoanisole to mutagenic products. 1-Napthylamine, 2-aminoanthracene, 2-acetylaminofluorene, and 2,4-diaminoanisole were negative or weakly mutagenic. N-Nitrosodimethylamine, N-nitrosomorpholine, the pesticide Aminocarb, and di(2-ethylhexyl)phthalate were not activated to mutagenic products by the ram seminal vesicle microsomal fraction.     

Characterization of potentially mutagenic products from the nitrosation of piperine.

Piperine is the main pungent principle of pepper, a spice consumed by people all over the world. It is the trans-trans isomer of 1-piperoylpiperidine and contains the methylene dioxy moiety. It is known to give unidentified mutagenic products on reaction with nitrite. The nitrosation reaction of piperine is of concern as endogenous nitrosation could take place in the human stomach from ingested precursors, piperine and nitrite. Nitrites can be ingested directly by consuming cured foods or indirectly as nitrates, which could be converted to nitrites under appropriate conditions. We have nitrosated piperine using aqueous nitrous acid and have isolated and identified some N-nitroso and C-nitro compounds. Their isolation, characterization and potential mutagenicity has been discussed.     

Chemical and mutagenic properties of alpha-phosphonooxynitrosamines

The chemical and mutagenic properties of the products of solvolysis of alpha-acetoxynitrosamines in phosphate buffer were investigated. alpha-Acetoxynitrosamines decomposed in two ways: O-acyl fission yielded alpha-hydroxynitrosamines, which decomposed into aldehydes and alcohols, while O-alkyl fission gave a resonance hybrid of alpha-N-nitrosocarbonium and -iminium ions, which was trapped with phosphate and afforded alpha-phosphonooxynitrosamine. Formation of alpha-phosphonooxynitrosamines was dependent on the structure of alpha-acetoxynitrosamines; those with a secondary alpha-phosphonooxy group, including cyclic nitrosamines, were easily formed, while among those with a primary phosphonooxymethyl group, only those with an alkyl group containing a branched alpha-carbon as isopropyl, sec-butyl and tert-butyl were isolated. They were good substrates of alkaline phosphatase and showed a nuclear magnetic resonance spectrum due to the presence of a phosphorus atom. They were decomposed by acid catalysis, and the rate was dependent on the structure. They were directly mutagenic in bacterial tester strains, except for a compound with a tert-butyl group. The activity was similar or stronger in Salmonella typhimurium TA1535 and much weaker in Escherichia coli WP2 and WP2 hcr- than those of alpha-acetoxynitrosamines. Stability in neutral aqueous solution and the strong mutagenicity of alpha-phosphonooxynitrosamines suggested their possible involvement in metabolic activation as a precursor of alpha-hydroxynitrosamines, and also in the organotropic carcinogenicity of N-nitrosodialkylamines as a transport form.     

Acceleration of nitrosamine formation at pH 3.5 by microorganisms.

Rate enhancements of from 12 to 49-fold occurred when dihexylamine was nitrosated at pH 3.5 in the presence of bacteria and yeast cells at a concentration of 12 mg/ml. Rates were similar in the presence of either boiled or unheated cells. The magnitude of the rate enhancement for nitrosation of other amines depended on the alkyl chain length. A nonenzymatic mechanism involving hydrophobic interactions of the precursor amines and cellular constituents is proposed.     

Production of unsaturated carbonyl compounds during metabolism of hydroperoxy fatty acids by colonic homogenates

Recent evidence has suggested that unsaturated carbonyl compounds may be the ultimate mitogens produced from the primary auto-oxidation products of unsaturated fatty acids. The present study has investigated the metabolism of 13-hydroperoxyoctadecandienoic acid (13-ROOH) by rat colon homogenates. This hydroperoxide is one of the primary products formed from the oxygenation of linoleic acid, the most abundant dietary polyunsaturated fatty acid. Incubation mixtures contained [1-14C]13-ROOH and colonic homogenates prepared from male Sprague-Dawley rats. After 30 min of incubation the reaction was quenched and the products extracted for analysis by HPLC. The identity of the eluted products were verified by UV, MS and NMR spectroscopy. The major products include a mixture of isomers of 2,4-dienone C18 fatty acids and 13-hydroxyoctadecadienoic acid. Direct comparison of homogenate metabolism to the hematin-catalyzed, alkoxyl radical-mediated decomposition of 13-ROOH shows some significant differences. In particular, no epoxy products are detected in the presence of tissue homogenates whereas these are the major products observed during the decomposition of 13-ROOH by hematin and a number of other agents. These experiments demonstrate the production of relatively large amounts of unsaturated carbonyl-containing fatty acids during the metabolism of hydroperoxy fatty acids by colonic tissue. The major product, 13-oxo-9Z,11E-octadecadienoic acid, when instilled intrarectally stimulates the incorporation of [3H]deoxythymidine into colonic mucosal DNA, and induces colonic mucosal ornithine decarboxylase activity in vivo. These findings have important implications for the mechanism by which dietary fat promotes colon tumorigenesis as the formation of relatively reactive 2,4-dienones may be a key to the in vivo mitogenic activity of oxidized fatty acids.