Mutation Research 443Ž. 1999 129±138 www.elsevier.comrlocatergentox Community address: www.elsevier.comrlocatermutres N-Nitroso compounds in the diet William Lijinsky 11398 High Hay DriÕe, Columbia, MD 21044, USA Received 7 October 1998; received in revised form 12 February 1999; accepted 12 February 1999 Abstract N-Nitroso compounds were known almost 40 years ago to be present in food treated with sodium nitrite, which made fish meal hepatotoxic to animals through formation of nitrosodimethylamineŽ. NDMA . Since that time, N-nitroso compounds have been shown in animal experiments to be the most broadly acting and the most potent group of carcinogens. The key role of nitrite and nitrogen oxides in forming N-nitroso compounds by interaction with secondary and tertiary amino compounds has led to the examination worldwide of foods for the presence of N-nitroso compounds, which have been found almost exclusively in those foods containing nitrite or which have become exposed to nitrogen oxides. Among these are cured meats, especially baconÐand especially when cooked; concentrations of 100 mgkgy1 have been found or, more usually, near 10 mgkgy1. This would correspond to consumption of 1 mg of NDMA in a 100-g portion. Much higher concentrations of NDMAŽ. but lower ones of other nitrosamines have been found in Japanese smoked and cured fish Ž more than 100 mg kgy1. Beer is one source of NDMA, in which as much as 70 mg ly1 has been reported in some types of German beer, although usual levels are much lowerŽ 10 or 5 mg ly1. ; this could mean a considerable intake for a heavy beer drinker of several liters per day. Levels of nitrosamines have been declining during the past three decades, concurrent with a lowering of the nitrite used in food and greater control of exposure of malt to nitrogen oxides in beer making. There have been declines of N-nitroso compound concentrations in many foods during the past two decades. The small amounts of nitrosamines in food are nonetheless significant because of the possibilityÐeven likelihoodÐthat humans are more sensitive to these carcinogens than are laboratory rodents. Although it is probable that alkylnitrosamidesŽ which induce brain tumors in rodents. are present in cured meats and other potentially nitrosated products in spite of much searching, there has been only limited indirect evidence of their presence. q 1999 Elsevier Science B.V. All rights reserved. Keywords: N-Nitroso; Diet; Nitrosodimethylamine 1. Introduction recent and are distinguished by being very potent. They are seldom present by deliberateŽ if coinciden- In their examination of the causes of human tal. addition. Since N-nitroso compounds are easily cancer, Doll and Petowx 1 ascribe a major roleŽ. 60% formed by interaction of a secondary amino com- to `diet', based on epidemiological observations; they pound with a nitrosating agentŽ commonly a nitrite did not particularize which agents in the food might salt, but also `nitrous gases'. , it might have been be responsible, or those that might be more impor- expected that foods treated with nitrites for colour- tant than others. Among the numerous chemical ing, flavouring and preservation would likely contain carcinogens that have been detected in human food N-nitroso compounds, but this apparently was not and drink, N-nitroso compounds are among the most thought of. 1383-5718r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S1383-5742Ž. 99 00015-0 130 W. LijinskyrMutation Research 443() 1999 129±138 The first inkling of such a connection was early in so, since the size of the dose, the frequency of the the 1960s when some sheep in Norway died of liver dose and the route of administration to a certain toxicity after feeding on fish meal that had been speciesŽ. e.g., rats can change the affected organ treated with sodium nitritewx 2±4 . It would not have from lung to kidney to liver, even changing the been expected that a nitrosamine, nitrosodimeth- target cell from which tumors arise from hepatocytes ylamineŽ. NMDA , would be present in the nitrite- to endothelial cellswx 11 . These modulations make it treated fish meal, since nitrosation of aminesŽ in this difficultÐif not impossibleÐto predict, based on case, dimethylamine and trimethylamine. was con- experiments in rats or mice, which would be the sidered to take place only in acid solution, and the target organ of a particular N-nitroso compound in fish meal milieu was neutral or alkaline. Much later, humansŽ. or, in fact, in any other species . On the Keefer and Rollerwx 5 demonstrated that interaction other hand, NDEA has induced liver tumors in most of a secondary amine with nitrite readily took place of the species in which it has been tested, including in alkaline medium in the presence of a carbonyl rodents, snakes, birds, molluscs and monkeyswx 10,12 . compoundŽ especially an aldehyde±formaldehyde in Of the several hundred N-nitroso compounds that the case of fish meal. The presence of considerable have been examined, only a few are likely to be quantities of nitrosamines in cured tobacco is also a encountered by humans outside the laboratory and consequence of nitrosation in non-acid mediumwx 6 . almost all are nitrosamines, which are stable and not It is noteworthy that the amount of NMDA in the directly acting. As a group, nitrosamines induce fish meal was large, even considering the potent tumors in a variety of organswx 13 , including liver, hepatotoxicity of NDMA, because some of the sheep lung, kidney, bladder, pancreas, esophagus and died. tongueÐdepending on the speciesÐbut not in skin, These observations followed closely the first re- brain, colon or bone. On the other hand, the unstable port of toxicity and carcinogenicity of NDMA by and directly acting nitrosamidesŽ alkylnitrosoureas or Magee and Barneswx 7 , after which there was an alkylnitrosocarbamates. induce tumors of the ner- explosion of interest in the toxicology of the N- vous systemŽ. in rats but not in hamsters , stomach, nitroso compounds, which are among the simplest of gastrointestinal tract and bone. Many of the tumors chemical carcinogens. Most of the experiments done induced by N-nitroso compounds are similar to the subsequently involved the testing of N-nitroso com- analogous human tumors and it is tempting to con- pounds of various structuresŽ several hundred com- clude that the latter might be caused by human pounds. , the results of which provided considerable exposure to N-nitroso compounds. Such human ex- insight into the mechanisms by which N-nitroso posures occur in certain work environments, as a compounds were activated and induced cancerwx 8,9 . consequence of some habitsŽ smoking and chewing Among the findings were that 40 or more species tobacco. , in the use of particular household products that have been treated with, e.g., nitrosodiethylamine Ž.cosmetics and shampoos , but probably the most Ž.NDEA , were all susceptible to its carcinogenic widespread exposure is in food and drink. A related actionwx 10 , suggesting strongly that, unlike other exposureŽ. which will not be gone into here is types of carcinogen, there was probably no non-sus- through the formation of N-nitroso compounds by ceptible speciesŽ. e.g., humans . Another finding was interaction in the stomach of secondary and tertiary that many species differed as to which organŽ. s amino compoundsŽ. in food or medicines with nitrite responded to a particular N-nitroso compound, which Ž.in food and in saliva or other nitrosating agents. also depended on the chemical structure of the car- This endogenous nitrosation, probably an important cinogen. There were some N-nitroso compounds that source of N-nitroso compounds, has been exten- appeared to be inactiveŽ. i.e., non-carcinogenic , sively discussed for many yearswx 14 and has been which also gave insight into mechanisms of carcino- demonstrated experimentallywx 15±17 . It has also genesis by these compounds. It would, however, be been studied epidemiologically in connection with misleading to claim that the mechanism by which human cancerwx 18 . Most human cancers, with the any N-nitroso compound induces tumors is under- exception of those caused by tobacco use, have no stoodŽ. or, indeed, any other carcinogen , particularly known cause, and widespread exposure to N-nitroso W. LijinskyrMutation Research 443() 1999 129±138 131 compounds in food provides one link that should not methylnitroso-benzylamine in some foods in China be ignored. that were associated with a high incidence of esophageal cancer in inhabitants of those areaswx 21 ; these claims were, in some instances, later shown to 2. Methods of analysis for N-nitroso compounds be inaccuratewx 22 . A major deficiency in our information about the At a meeting in Jamaica in 1968 sponsored by the distribution of N-nitroso compounds in foods is the World Health Organization dealing with distribution carcinogenic non-volatile compounds. These com- and occurrence of carcinogens, it was concluded prise two important groups, the alkylnitrosamides that, while trace analysis of polynuclear hydrocar- Ž.including alkylnitrosoureas and hydroxylated ni- bons was possible to hundredths of a part per million trosaminesw including nitrosodiethanolamine and ni- or less, detection of nitrosamines was unlikely to be trosobis-Ž. 2-hydroxypropyl aminex . These are impor- feasible even at 1 ppm, except in unusual circum- tant carcinogens of considerable potency and can be stances. At about the same time, there was under isolated chromatographically only by high-pressure development by the Thermo-Electron Corporation liquid chromatographyŽ. HPLC , not gas chromatog- Ž.ŽWaltham, Massachusetts, USA , a device thermal raphy; identification at low concentrations of alkylni- energy analyzer or TEA. for detecting N-nitroso trosamides is not easy, because they are not very compoundsŽ. although not entirely specific to them stablewx 23 , but they are important because they are using the electronic measurement of the infrared among the few carcinogens that induce tumors of the light emitted by NO liberated by pyrolysis of nitroso nervous system in experimental animalswx 24 and are compounds.