A-nitrosamines formation

Mills AL, M Alexander (1976) A-nitrosamine formation by cultures of several microorganisms. Appl Environ Microbiol 31 892-895. 

In the presence of a nucleophilic anion such as I, Br, CP, SCN, acetate or phthalate, nitrous acid can be converted into more active nitrosating species. A -nitrosamines formation can be accelerated by certain microorganisms at acid pH values. On the other hand, the nitrosation reactions can be inhibited by compounds such as ascorbic acid, sulfamic acid, tocopherol, and others. °... 

The A-nitrosamine formation in tobacco smoke is determined by the nitrate content of the tobacco... More importantly... selective removal (70 to 80 percent) of volatile nitrosamines from the smoke can be achieved by cellulose filters [sic] ... At present, it has not been demonstrated that a significant reduction of volatile A-nitrosamines will lead to a significant reduction of the tumorigenic potential of cigarette smoke. 

Chamberlain, W.J., J.L. Baker, O.T. Chortyk, and M.G. Stephenson A-Nitrosamine formation in tobacco 39th Tobacco Chemists Research Conference, Program Booklet and Abstracts, Vol. 39, Paper No. 23, 1985, p. 

Theiler, R., K. Sato, T. Aspelund, and A. Miller Inhibition of A-nitrosamine formation in a cured ground pork belly model system J. Food Sci. 49 (1984) 341-344. Thompson, H.C. Jr, S.M. BUledeau, B.J. Miller, E.B. Hansen Jr, J.P. Freeman, and M.L. Wind Determination of A-nitrosamines and A-nitrosamine precursors in rubber nipples from baby pacifiers by gas chromatography thermal energy analysis J. Toxicol. Env. Hlth. 13 (1984) 615-632. 

The content of free diethanolamine in particular is controlled in these products because secondary amines open the possibility of A-nitrosamine formation if they should come in contact with nitrites. The methods most frequently used nowadays for alkanolamine analysis are gas chromatography, ion chromatography, and capillary electrophoresis. The titration methods have become less useful over the last decade as the concentration of the free alkanolamines in commercial products continues to decline. 

Inhibition of nitrosation is generally accompHshed by substances that compete effectively for the active nitrosating iatermediate. /V-Nitrosamine formation in vitro can be inhibited by ascorbic acid [50-81-7] (vitamin C) and a-tocopherol [59-02-9] (vitamin E) (61,62), as well as by several other classes of compounds including pyrroles, phenols, and a2iridines (63—65). Inhibition of iatragastric nitrosation ia humans by ascorbic acid and by foods such as fmit and vegetable juices or food extracts has been reported ia several instances (26,66,67). 

Mutagenicity. The AJ-nitrosamines, in general, induce mutations in standard bacterial-tester strains (117). As with carcinogenicity, enzymatic activation, typically with Hver microsomal preparations, is required. Certain substituted A/-nitrosamine derivatives (12) induce mutations without microsomal activation (31,33,34). Because the a-acetoxy derivatives can hydroly2e to the corresponding a-hydroxy compounds, this is consistent with the hypothesis that enzymatic oxidation leads to the formation of such unstable a-hydroxy intermediates (13) (118). However, for simple /V-nitrosamines, no systematic relationship has been found between carcinogenicity and mutagenicity (117,119—123). 

Biochemical Functions. Ascorbic acid has various biochemical functions, involving, for example, coUagen synthesis, immune function, dmg metabohsm, folate metaboHsm, cholesterol cataboHsm, iron metaboHsm, and carnitine biosynthesis. Clear-cut evidence for its biochemical role is available only with respect to coUagen biosynthesis (hydroxylation of prolin and lysine). In addition, ascorbic acid can act as a reducing agent and as an effective antioxidant. Ascorbic acid also interferes with nitrosamine formation by reacting direcdy with nitrites, and consequently may potentially reduce cancer risk. 

Inhibition of Nitrosamine Formation. Nitrites can react with secondary amines and A/-substituted amides under the acidic conditions of the stomach to form /V-nitrosamines and A/-nitrosamides. These compounds are collectively called N-nitroso compounds. There is strong circumstantial evidence that in vivo A/-nitroso compounds production contributes to the etiology of cancer of the stomach (135,136), esophagus (136,137), and nasopharynx (136,138). Ascorbic acid consumption is negatively correlated with the incidence of these cancers, due to ascorbic acid inhibition of in vivo A/-nitroso compound formation (139). The concentration of A/-nitroso compounds formed in the stomach depends on the nitrate and nitrite intake. 

In view of its potential for nitrosamine formation, a more detailed knowledge of the atmospheric reactions and products of UDMH is clearly desirable. In order to provide such data for UDMH and other hydrazines we have studied their dark reactions in air, with and without added O3 or NO, and have investigated their atmospheric photooxidation in the presence of NO ( 9 ). In this paper, we report the results we have obtained to date for UDMH. 

The results of the study reported here show clearly that, upon release into the atmosphere, N,N-dlmethylhydrazlne (UDMH) can be rapidly converted to N-nitrosodimethylamine by its reaction with atmospheric ozone. A similar conclusion can be reached concerning nitrosamine formation from other unsymmetrically disubstituted hydrazines ... 

Under these conditions, although nitrosamine formation appears to occur to some extent, formation of an unknown product (or set of products) is also observed. On the basis of mechanistic considerations we believe this product to be primarily a nitroso-hydrazine. Upon photolysis, this compound may give rise to an N-nitrohydrazine, or, when O3 is present, to the nitrosamine. 

A research program in progress at Raltech Scientific Services is designed to find inhibitors which will prevent nitrosamine formation in cosmetic products, A review of the literature (4) indicated that the oil phase of emulsions may play an important role in nitrosation chemistry. Thus, results from studies in water alone could be misleading when reduced to practice. 

Evidence exists that the relative solubility of amines and inhibitors in heterogeneous oil-water systems could be decisive in formation of nitrosamines and blocking these reactions, Nitrosopyrrolidine formation in bacon predominates in the adipose tissue despite the fact that its precursor, proline, predominates in the lean tissue (5,6,7). Mottram and Patterson (8) partly attribute this phenomenon to the fact that the adipose tissue furnishes a medium in which nitrosation is favored, Massey, et al, (9) found that the presence of decane in a model heterogeneous system caused a 20-fold increase in rate of nitrosamine formation from lipophilic dihexylamine, but had no effect on nitrosation of hydrophilic pyrrolidine. Ascorbic acid in the presence of decane enhanced the synthesis of nitrosamines from lipophilic amines, but had no effect on nitrosation of pyrrolidine. The oil-soluble inhibitor ascorbyl palmitate had little influence on the formation of nitrosamines in the presence or absence of decane. 

In order to define the conditions of the growing cultures, buffered medium (VL) inoculated with E, coli ATCC 11775 and supplemented with nitrate, glucose and DMA was incubated at 37 C, and pH, nitrite concentration, nitrate concentration, cell growth and nitrosamine formation were followed (Fig. 1). Within 2 hrs, >90% of the nitrate is converted to nitrite (some of the nitrite is further reduced) and over 8 hrs the pH drops from 7.3 to 6.0. This would indicate that in experiments carried out for 20 hrs or more the control medium should be adjusted to pH 6.0 to 6.5 and nitrite should be added rather than nitrate. Such a control medium (VL) was supplemented with nitrite and DMA and NDMA formation was followed (Fig. 2). It can be seen that even without the addition of cells the rate of nitrosation is 4 fold greater than... 

N-Nitrosamine inhibitors Ascorbic acid and its derivatives, andDC-tocopherol have been widely studied as inhibitors of the N-nitrosation reactions in bacon (33,48-51). The effect of sodium ascorbate on NPYR formation is variable, complete inhibition is not achieved, and although results indicate lower levels of NPYR in ascorbate-containing bacon, there are examples of increases (52). Recently, it has been concluded (29) that the essential but probably not the only requirement for a potential anti-N-nitrosamine agent in bacon are its (a) ability to trap NO radicals, (b) lipophilicity, (c) non-steam volatility and (d) heat stability up to 174 C (maximum frying temperature). These appear important requirements since the precursors of NPYR have been associated with bacon adipose tissue (15). Consequently, ascorbyl paImitate has been found to be more effective than sodium ascorbate in reducing N-nitrosamine formation (33), while long chain acetals of ascorbic acid, when used at the 500 and lOOO mg/kg levels have been reported to be capable of reducing the formation of N-nitrosamines in the cooked-out fat by 92 and 97%, respectively (49). 

Commercially available nonfat dried milk and dried buttermilk have also been shown to contain small but detectable levels of NDMA (, , ). It has been suggested that N-nitrosamine formation is possible in foods that are dried in a direct-fired dryer (65). In such a dryer, the products of combustion come into direct contact with the food being dried, and N-nitrosamine formation is probably due to the reaction between secondary and/or tertiary amines in the food and the oxides of nitrogen that are produced during fuel combusion (65). 

In classical organic chemistry, nltrosamlnes were considered only as the reaction products of secondary amines with an acidified solution of a nitrite salt or ester. Today, it is recognized that nitrosamines can be produced from primary, secondary, and tertiary amines, and nltrosamides from secondary amides. Douglass et al. (34) have published a good review of nitrosamine formation. For the purposes of this presentation, it will suffice to say that amine and amide precursors for nitrosation reactions to form N-nitroso compounds are indeed ubiquitous in our food supply, environment, and par-... 

Indeed, given an improperly designed or understood system, a blocking agent, like ascorbic acid, could be catalytic toward nitrosamine formation. For example, if the source of nitrosatlng agent is nitrite ion and the susceptible amine is in the lipid phase, conceivably ascorbic acid could cause the rapid reduction of nitrite ion to nitric oxide which could migrate to the lipid phase. Subsequent oxidation of NO to NO in the lipid phase could cause nitrosation. 

If the concepts and facts presented in this paper are correct, a major kind of human cancer in many regions of the world, cancer of the stomach, is due to a type of nitroso compound, a nitrosoureido derivative, even though not a nitrosamine It is quite certain that the formation of such compounds can be blocked by vitamin C and vitamin E, as well as by some other substances such as gallates Thus, the primary prevention of cancer caused by nitroso compounds is readily accomplished through an adequate Intake of such harmless inhibitors with every meal from infancy onwards ... 

Reliable analytical methods are available for determination of many volatile nitrosamines at concentrations of 0.1 to 10 ppb in a variety of environmental and biological samples. Most methods employ distillation, extraction, an optional cleanup step, concentration, and final separation by gas chromatography (GC). Use of the highly specific Thermal Energy Analyzer (TEA) as a GC detector affords simplification of sample handling and cleanup without sacrifice of selectivity or sensitivity. Mass spectrometry (MS) is usually employed to confirm the identity of nitrosamines. Utilization of the mass spectrometer s capability to provide quantitative data affords additional confirmatory evidence and quantitative confirmation should be a required criterion of environmental sample analysis. Artifactual formation of nitrosamines continues to be a problem, especially at low levels (0.1 to 1 ppb), and precautions must be taken, such as addition of sulfamic acid or other nitrosation inhibitors. The efficacy of measures for prevention of artifactual nitrosamine formation should be evaluated in each type of sample examined. 

Major emphasis in studies of N-nitroso compounds in foods has been placed upon volatile nitrosamines, in part because these compounds are relatively easy to isolate from complex matrices by virtue of their volatility. Procedures utilizing atmospheric pressure or vacuum distillation have been used by most investigators, with variations of the method of Fine e al. (2) being among the most popular. This procedure employs vacuum distillation of a mineral oil suspension of the sample with optional addition of water to improve nitrosamine recovery from low moisture content samples (6) The usual approach to prevention of nitrosamine formation during analysis involves adding sulfamic acid or ascorbate to destroy residual nitrite at an early stage of sample preparation. 

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