Proceedings ofthe Nutrition Society (1.985)~ 44, (95-100
`
`95
`
`THE BRITISH SOCIETY OF GASTROENTEROLOGY/
`NUTRITION SOCIETY/GLAXO INTERNATIONAL TEACHING DAY
`
`Nutrition and nitrosamine formation
`
`By B. C. CHALLIS, Department of Chemistry, Imperial College of Science and
`Technology, South Kensington, London S W7 d Z
`
`Nutrition and the fmmation of N-nitroso compounds
`N-nitroso compounds are an important group of chemical carcinogens that could
`be involved in human cancer (Magee, 1982). Only a few examples have been found
`in food and beverages (Assembly of Life Sciences, 1981) but suitable precursors are
`common dietary constituents (Assembly of Life Sciences, 1981), and the mildly
`acidic conditions of the stomach are favourable for their formation (Challis, 1981).
`Over 90% of the N-nitroso compounds tested have proven to be carcinogenic,
`some in as many as thirty species ranging from mice to primates (Magee et al.
`1976). Many are organ-specific carcinogens, producing tumours remote from the
`site of their administration (Magee et al. I 976). Nonetheless, unequivocal evidence
`that N-nitroso compounds cause human cancer has proven elusive.
`
`Formation of N-nitroso compounds
`N-nitroso compounds are usually formed by the interaction of a nitrosating
`agent (NOX) derived from either nitrite salts or nitrogen oxides with an amino
`substance (e.g. R,NH) as in eqn (I). The amino substance can be one of several
`R,NH + NOX s R,NNO + HX
`types of compound such as secondary and tertiary amines, secondary and tertiary
`amides, peptides, substituted ureas, guanidines and urethanes. In the case of
`amines, amides and ureas, the products are the N-nitrosamines, N-nitrosarnides
`and N-nitrosoureas shown in Fig. I. The mechanism and extent of formation of the
`N-nitroso derivative is dependent on both the structure of the amino substance and
`the source of the nitrosating agent, as well as the reaction conditions.
`With nitrite salts, the most extensive reactions apply to weakly basic amino
`substances (e.g. aromatic amines, amides, ureas) under aqueous acidic conditions
`(Challis, 1981). For secondary amines and amino acids, formation of their
`N-nitroso derivatives usually passes through a maximum at pH 2.5-3'5 where the
`yield is inversely proportional to the basicity of the amino substance (Mirvish,
`1975). These reactions are catalysed by substances such as halides (Douglass et al.
`
`(1)
`
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`Human Power of N Company
`EX1031
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`

`

`B. C. CHALLIS
`
`I985
`
`(b)
`
`(4
`RNH-C ,
`
`0
`//
`
`96
`
`(a>
`
`, N-NO
`
`R-C ,
`
`0
`4
`
`R \
`N-NO
`N-NO
`/
`/
`R'
`R'
`R'
`N-Nitrosamine
`N-Nitrosamide
`N-Nitrosourea
`Fig. I . N-nitroso compounds formed from ( a ) amines, ( b ) amides and (c) ureas. R, R' = alkyl, aryl
`or part of a cyclic structure.
`
`1978), thiocyanate (Douglas et al. 1978), and formaldehyde (Casado et al. 1981).
`Tertiary amines react similarly to secondary amines, but usually to a much lower
`extent (Mirvish, 1975; Gowenlock et al. 1979). For amides, peptides, ureas and
`guanidines formation of their N-nitroso derivatives rarely occurs above pH 4, but
`increases steadily with increasing acidity (i.e. decreasing pH) (Mirvish, 1975;
`Challis et al. 1985). These reactions are not catalysed by other substances and
`their extent varies in a complex way with substrate structure and basicity
`(Mirvish, 1975; Challis, 1981).
`With nitrogen oxides, reactions are most extensive for strongly basic amino
`substances (e.g. aliphatic and heterocyclic amines) under non-aqueous or neutral
`and alkaline aqueous conditions (Challis & Kyrtopoulos, 1979). Oxygen (Challis &
`Kyrtopoulos, 1979)~ iodine (Challis 8~ Outram, 1979), polyhydroxy compounds
`(e.g. carbohydrates) (Challis & Shuker, 1980) and several metal salts (Challis et al.
`1978; Challis & Outram, 1978) have an enhancing effect, whereas acidic conditions
`are inhibitory (Challis & Kyrtopoulos, 1979). Amides, peptides, ureas and
`guanidines do not react with nitrogen oxides in aqueous media (Challis &
`Kyrtopoulos, 1979).
`Many compounds and components of foods other than amino substances can
`react with nitrite salts and nitrogen oxides, and thus reduce the amounts available
`for the formation of N-nitroso compounds. Such compounds include ascorbic acid
`(vitamin C) (Mirvish, 1981), a-tocopherol (vitamin E) (Newmark & Mergens, 1981)
`and other natural and synthetic antioxidants (e.g. tannins, chlorogenic acid,
`butylated hydroxy anisole, sulphite) (Douglas et al. 1978; Challis, 1981; Mirvish
`1981). These either bind the nitrosating agent (NOX) irreversibly or reduce it to
`nitric oxide (NO)-a
`relativly ineffectual nitrosating agent-as
`shown for
`ascorbic acid in eqn (2).
`
`HO 3
`
`
`
`0OH
`
`+
`
`2 N O X
`
`
`--f HO & 0 + 2 N 0 + H 2 0 OH
`
`(2)
`
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`

`VOl. 44
`International Teaching Day
`97
`Further, primary amines, most amino acids and peptides may undergo
`deamination (eqn (3)) (Ridd, 1961; Challis, 1981), which also reduces the amount
`of nitrosating agent without forming N-nitroso compounds.
`RX + N, + H,O
`RNH, + NOX
`
`--f
`
`(3)
`
`Dieta y occurrence of N-nitroso compounds
`Combustion generates nitrogen oxides and nitrite salts are added as a
`preservative to some foods. These can generate N-nitroso compounds during
`processing and cooking if conditions allow the nitrosating agents to interact with
`amino substances in the foodstuff. Low levels of volatile N-nitrosamines have been
`found in some foods and beverages (e.g. N-nitrosodimethylamine in beer
`(Assembly of Life Sciences, 1981 ; Spiegelhalder et al. 1979) and N-nitrosopyrroli-
`dine in bacon (Assembly of Life Sciences, 1981)). Where such contaminants have
`been identified, changes in processing technology have reduced their levels
`(Assembly of Life Sciences, 1981; Preussmann et al. 1981). It is conceivable,
`however, that some foods and beverages contain higher levels of largely
`unidentified non-volatile N-nitroso compounds (Pollock, I 98 I). Their identi-
`fication, origin and health risk are the subject of much current work.
`
`Dieta y occurrence of precursors
`From a nutritional standpoint, the occurrence of precursors is of as much
`interest as N-nitroso compounds because the acidic conditions of the stomach are
`suitable for nitrosation reactions.
`Amino substances of one sort or another are present in most foods. This
`information has been reviewed (Singer & Lijinsky, 1976; Neurath et al. 1977;
`Maga, 1978; Smith, 1981), but much is either too qualitative or incomplete for
`reliable health-risk assessment. Individual secondary and tertiary amines in most
`foods rarely exceed 10 ppm and more often are less that 2 pprn (Singer & Lijinsky,
`1976; Neurath et al. 1977; Maga, 1978; Smith, 1981). One exception is fish, where
`up to 740 pprn dimethylamine (Singer & Lijinsky, 1976) and 1400 pprn
`trimethylamine (Maga, 1978) have been reported in some samples. The highest
`concentrations, however, may relate to spoilage (Gruger, 1972). Cocoa products
`and cheeses are usually rich in amines (Neurath et al. 1977; Maga, 1978; Smith,
`1981), but most are primary compounds which should undergo deamination (eqn
`(3)) and therefore reduce the overall formation of N-nitroso compounds. Further,
`secondary and tertiary amines in foods are often associated with 1000 fold larger
`concentrations of ammonia (Neurath et al. 1977; Maga, 1978; Smith, 1981), which
`should also decompose nitrite and nitrogen oxides without the formation of
`N-nitroso compounds. Less information is available about the presence of amides,
`ureas and guanidines in the diet, despite forceful arguments that their N-nitroso
`derivatives could be especially important in gastric cancers (Mirvish, 1983).
`Alkylureas and alkylguanidines have been found in some fish and meat products
`(Fujinaka et al. 1976; Mirvish et al. 1980), and in fried bacon (Mirvish et al. 1980),
`
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`

`B. C. CHALLIS
`I985
`98
`probably from the oxidation of creatine and creatinine. Further, proteins and
`peptides are the most abundant dietary nitrogen constituents and their behaviour
`towards nitrosating agents should be similar to amides and ureas (Challis, 1981).
`Until recently (Challis et al. 1985), their nitrosation and the chemical and
`biological properties of their N-nitroso derivatives have been largely overlooked.
`Because nitrate is reduced to nitrite by oral and gastric bacteria (Ralt &
`Tannenbaum, 1981) the dietary intake of both is relevant to gastric nitrosation.
`Both are present in food, especially vegetables (White, 1975; Assembly of Life
`Sciences, 1981), and in most water supplies at low level (Assembly of Life
`Sciences, 1981). Both form in the lungs on inhalation of the nitrogen oxides in
`polluted atmospheres and in tobacco smoke (Goldstein et al. 1980; Oda et ul.
`1981). Nitrite is added to some meats as a preservative against Clostridium
`botulinum (Assembly of Life Sciences, 1981). Thus, ingestion of nitrite and nitrate
`salts is unavoidable. A recent estimate puts the USA dietary nitrite intake at
`approximately 0 . 8 mg/d of which 4070 derives from cured meat, 3570 from cereal
`products and 1570 from vegetables, and the dietary nitrate intake at approximately
`75 mg/d of which 85% is from vegetables (Assembly of Life Sciences, 1981).
`Further, these intakes may be increasing in consort with the use of artificial
`fertilizers, which raise the concentration of nitrite and nitrate salts in both
`vegetables (White, 1975; Assembly of Life Sciences, 1981) and water supplies
`(Assembly of Life Sciences, 198 I). Vegetables, however, often contain
`correspondingly high levels of antioxidants (e.g. ascorbic acid and polyphenols)
`(Assembly of Life Sciences, 1981; Fine et al. 1982) which are able to react with
`nitrosating agents (eqn (2)) and therefore reduce the formation of N-nitroso
`compounds.
`
`Gastric nitrosation
`The acidic conditions of the stomach are suitable for nitrosation by nitrite from
`food and swallowed saliva. About 25% of ingested nitrate is recirculated into the
`saliva of which about 20% (i.e. 5% of the ingested nitrate) is then reduced to
`nitrite by oral bacteria (Spiegelhalder et ul. 1976; Tannenbaum et al. 1976). Of the
`nitrite entering the stomach, about 20% arises directly from food and about 80%
`from the reduction of salivary nitrate. Thus, gastric nitrosation is more dependent
`on nitrate than nitrite intake.
`Ingestion of proline and vegetable juices rich in nitrate increases urinary yields
`of N-nitrosoproline (eqn (4)) (Ohshima & Bartsch, 1981), and human gastric
`aspirates contain small amounts of largely unidentified N-nitroso compounds
`(Reed et al. 1981; Milton-Thompson et al. 1982). This is good evidence that
`humans allow the reduction of nitrate to nitrite and sustain gastric nitrosation
`
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`

`99
`International Teaching Day
`VOl. 44
`In healthy humans, gastric nitrite concentrations oscillate and reach maxima of
`about 30 PM (with considerable individual variation) 1-2 h after the consumption
`of food (Milton-Thompson et al. 1982). Gastric acidity varies similarly from about
`pH I on fasting to about pH 3 after food (Milton-Thompson et al. 1982). Under
`these conditions, N-nitroso compounds are most likely to form from aromatic
`amines, ureas and peptides (Challis et al, 1982). In patients who have pernicious
`anaemia (Ruddell et al. 1978), or have undergone gastric surgery (Schlag et al.
`1980), gastric pH is commonly above 5, nitrate-reducing bacteria multiply in the
`stomach and gastric nitrite concentrations are much higher. Whether these
`conditions produce enhanced levels of N-nitroso compounds is the subject of
`current debate.
`
`REFERENCES
`Assembly of Life Sciences (1981). The Health Effects of Nitrite, Nitrate and N-Nitroso
`Compounds Part r . Washington, DC: National Academy Press.
`Casado, J., Castro, A., Lopez-Quintella, M. A. & Vasquez Tato, J. (1981). Zeitschrifr f i r
`Physikalische Chemie, Neue Folge 127, 179-192.
`Challis, B. C. (1981). In Safety Evaluation of Nitrosatable Drugs and Chemicals, pp. 16-51 [G. C.
`Gibson and C. Ioannides, editors]. London: Taylor and Francis.
`Challis, B. C., Edwards, A., Hunma, R. R., Kyrtopoulos, S. A. & Outram, J. R. (1978). In
`Environmental Aspects of N-Nitroso Compounds, IARC Scientific Publication No. 19, pp.
`127-142 [E. A. Walker, M. Castegnaro, L. Griciute and R. E. Lyle, editors]. France: IARC.
`Challis, B. C., Hopkins, A. R., Milligan, J. R., Mitchell, R. C. & Massey, R. C. (1985). 8th
`International Meeting on N-Nitroso Compounds: Occurrence and Biological Effects, Banff,
`Canada, September 1983 (In the Press).
`Challis, B. C. & Kyrtopoulos, S. A. (1979). Journal of the Chemical Society Perkin Transactions
`11, 299-304.
`Challis, B. C., Lomas, S. J., Rzepa, H. S., Bavin, P. M. G., Darkin, S. W., Viney, N . J. & Moore,
`P. J. (1982) In Nitrosamines and Human Cancer, Banbuly Report no. 12, pp. 243-253 [P. N .
`Magee, editor]. New York: Cold Spring Harbor Laboratory.
`Challis, B. C. & Outram, J. R. (1978). Journal ofthe Chemical Society Chemical Communications
`707-708.
`Challis, B. C. & Outram, J. R. (1979). Journal of the Chemical Society Perkin Transactions II,
`2768-2775.
`Challis, B. C. & Shuker, D. E. G. (1980). Food and Cosmetic Toxicology 18,283-288.
`muglass, M. L., Kabacoff, B. L., Anderson, G. A. & Cheng, M. C. (1978). Journal ofthe Society
`of Cosmetic Chemists 29, 58 1-606.
`Fine, D. H., Challis, B. C., Hartman, P. & Van Ryzin, J. (1982). In N-Nitroso Compounds:
`Occurrence and Biological Effects, IARC Scientific Publication no. 41, pp. 379-396 [H.
`Bartsch, M. Castegnaro, I. K. O’Neill and M. Okado, editors]. France: IARC.
`Fujinaka, W., Masuda, Y. & Kuratsune, M. (1976). Gann 67,679-683.
`Goldstein, E., Goldstein, F., Peek, N. F. & Parks, N. J. (1980). In Nitrogen Orides and Their
`Effects on Health, pp. 143-160 [ S . D. Lee, editor]. Ann Arbor, Michigan: Ann Arbor Science.
`Gowenlock, B. G., Hutchinson, R. J., Little, J. & Pfab, J. (1979). Journal ofthe Chemical Society
`Perkin Transactions 11, I I 10-1 I 14.
`Gruger, E. H. (1972). Journal ofAgricultura1 and Food Chemistry 20, 781-785.
`Maga, J. A. (1978). CRC Critical Review of Food Science and Nutrition 10, 373-403.
`Magee, P. N. (editor) (1982). Nitrosamines and Human Cancer, Banbury Report no. 12. New
`York: Cold Spring Harbor Laboratory.
`In Chemical Carcinogens, ACS
`Magee, P. N., Montesano, R. & Preussmann, R.
`(1976).
`Monograph 173, pp. 491-625 [C. E. Searle, editor]. Washington, DC: American Chemical
`Society.
`
`Downloaded from https://www.cambridge.org/core. IP address: 38.113.135.42, on 03 Jun 2021 at 19:12:07, subject to the Cambridge Core terms of use, available
`at https://www.cambridge.org/core/terms. https://doi.org/10.1079/PNS19850015
`
`Page 5 of 6
`
`

`

`B. C. CHALLIS
`I985
`I00
`Milton-Thompson, G. J., Lightfoot, N. F., Ahmet, Z., Hunt, R. H., Barnard, J., Bavin, P. M. G.,
`Brimblecombe, R. W., Darkin, D. W., Moore, P. 1. & Viney, N. (1982). Lancet i, 1091-1095.
`Mirvish, S. S. (1975). Toxicology andApplied Pharmacology 31, 325-351.
`Mirvish, S. S. (1981). In Cancer 1980: Achievements, Challenges, Prospects for the 1980s, pp.
`557-587 [K. H. Burchenal and H. F. Oettgen, editors]. New York: Grune.
`Mirvish, S. S. (1983). Journal ofthe National Cancer Institute 71,6291647.
`Mirvish, S. S., Karlowski, K., Cairns, D. A., Sams, J. P., Abrahams, R. & Nielsen, J. (1980).
`Journal ofAgricultura1 and Food Chemistry 28, I 175-1 182.
`Neurath, G. B., Dunger, M., Pein, F. G., Ambrosius, D. & Schreiber, 0. (1977). Food and
`Cosmetic Toxicology 15,275-282.
`Newmark, H. L. & Mergens, W. J. (1981). In Inhibition of Tumour Induction and Development
`pp. 127-168 [M. Zedeck and M. Lipkin, editors]. New York: Plenum Press.
`Oda, H., Tsubone, H., Suzuki, T., Ichinose, T. & Kubota, K. (1981). Environmental Research 25,
`294-301.
`Ohshima, H. & Bartsch, H. (1981). Cancer Research 41,3658-3662.
`Pollock, J. R. A. (1981). Journal ofthe Institute ofBrewing 87, 356-359.
`Preussmann, R., Spiegelhalder, B. & Eisenbrand, G. (1981). In N-Nitroso Compounds. ACS
`Symposium Series 174, pp. 217-245 [R. A. Scanlan and S. R. Tannenbaum, editors].
`Washington, DC: American Chemical Society.
`Ralt, D. & Tannenbaum, S. R. (1981). In N-Nitroso Compounds, ACS Symposium Series 174, pp.
`157-164 [R. A. Scanlan and S. R. Tannenbaum, editors]. Washington, DC: American
`Chemical Society.
`Reed, P. I., Smith, P. L. R., Haines, K., House, F. R. & Walters, C. L. (1981). Lancet ii, 550-552.
`Ridd, J. H. (1961). Quarterly Review, Chemical Society, London 15,418-441.
`Ruddell, W. S., Bone, E. S., Hill, M. J. & Walters, C. L. (1978). Lancet i, 521-523.
`Schlag, P., Uhlrich, H., Merckle, P., Bockler, P., Peter, M. & Herfath, C. (1980). Lancet i,
`727-729.
`Singer, G . M. & Lijinsky, W. (1976). Journal ofAgricultura1 and Food Chemistry 24, 55-553.
`Smith, T. A. (1981). Food Chemistry 6, 169-200.
`Spiegelhalder, B., Eisenbrand, G. & Preussmann, R. (1976). Food and Cosmetic Toxicology 14,
`545-548.
`Spiegelhalder, B., Eisenbrand, G. & Preussmann, R. (1979). Food und Cosmetic Toxicology 17,
`29-3 I.
`Tannenbaum, S. R., Weisman, M. & Fett, D. (1976). Food and Cosmetic Toxicology 14,549-552.
`White, J. W. (1975). Journal ofAgricultural and Food Chemistry 23,886-891.
`
`Printed in Great Britain
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