N-Nitroso Compounds in the Diet

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.

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. Prior to this, analysis and detection of suspected of responsibility for brain tumors in chil- N-nitroso compounds involved working up a batch drenwx 18 . Methylnitrosourea has not been identified of material by preferential extraction, concentration directly in foodŽ. cured meats , but its presence has of the extract and chromatographic identification of been inferred by the identification of a methylated the N-nitroso compound in a particular band, often receptorŽ. nitroso-t-butylglycine formed by direct using a colour reagent. As mentioned, the sensitivity methylationwx 25 ; alkylnitrosamides are powerful, di- of these methods was not great, nor was the speci- rectly acting alkylating agents, which have been ficity. This resulted in many erroneous detections linked to their mutagenic and carcinogenic activities. Ž.and measurements of nitrosamines in cheeses, veg- Another group of non-volatile N-nitroso compounds, etable oils and other foods in which they were not almost all of which are non-carcinogenic or weakly present, or present at much lower concentrations than carcinogenic, is the nitrosamino acids, some of which those reported. In one series of experiments by this are quite widespread in foods. author, acetic acid was confused with NDMA because both had the same retention time on a particu- 3. Occurrence of N-nitroso compounds in food lar chromatographic column packing. The TEAwx 19 and beverages was not only more specific in detecting N-nitroso compoundsŽ a few other chemical types also pro- The use of sodium nitrite to colour, flavour and duced a response, but they were different in their preserveŽ. prevention of botulism meat and fish, behaviour in other respects, e.g., chromatographi- replacing sodium nitrate which had been used since callywx 20. but it was highly sensitive, enabling con- time immemorial, led to the search for N-nitroso centrations of fractions of a part per billion to be compounds in such foods, once nitrosamines were detected. Yet more recently, it has been required that classified as carcinogens in 1956wx 7 . Many investi- a chromatographic columnŽ gas±liquid or high-pres- gators in the field of food toxicology developed a sure liquid. be combined with a mass spectrometer great interest in this topic, as did the meat packing as detector to definitively identify a particular com- industry. There was a special interest in bacon, be- ponent as a N-nitroso compound. More recent data, cause it is so widely consumed. There developed in therefore, tend to be more reliable than earlier re- the 1970s quite a controversy over the use of nitrite ports, e.g., the large concentrations of NDEA and in bacon, which was often vacuum-packed with a 132 W. LijinskyrMutation Research 443() 1999 129±138 consequent risk of infection with the anaerobic Nitrosopyrrolidine was absent from uncooked bacon. Clostridium botulinum. The regulation thenŽ not al- The same was true of other cured meats; nitrosamino ways enforced. was that the maximum level of acids were present in the uncooked products, to- sodium nitrite in bacon should be 200 ppm; a similar gether with some moderate levels of nitrosamines regulation applied to other nitrite-cured meats in the Ž.mainly NDMA , but some nitrosopyrrolidine and USA, such as ham, sausagesŽ. many kinds , corned higher levels of NDMA in the cooked products. The beef, etc. Certain kinds of smoked fish, such as amount of nitrosopyrrolidine formed in cooked ba- smoked salmon, were also treated with sodium ni- con depends on the duration and temperature of trite, and the concentration was less well-regulated frying, much more at higher temperatures, very little than in meat. in microwave cooking. Another nitrosamine, nitroso- Analysis of bacon and other cured meats for thiazolidine, was discovered in bacon and other cured nitrosamines in the early yearsŽ. 1960s was not very meats, and appears to be derived from interaction of accurate nor specific. The most common N-nitroso cysteine, formaldehyde and nitrite. Nitrosothiazoli- compounds identified in foods were NDMA and dine was at first thought to be carcinogenic on the NDEA, but there were `identifications' of some ni- basis of analogy with the potent carcinogen, nitrosamines which were distinctly dubiouswx 26 . The trosooxazolidinewx 31 , and because some of its prepa- tendency was for investigators to examine the foods rations were mutagenic in the Ames test. Subse- in their own countries, so there is a wealth of quently, it was found that purified preparations of information about nitrosamine contamination world- nitrosothiazolidine were not mutagenic and a chronic wide, even if the accuracy of some of the reports is feeding study in rats was negativewx 32 , showing that suspected. its presence in cured meats was not a carcinogenic Because the isolation and identification of volatile risk. nitrosamines is relatively easy, most of the attention Recently, there has been some concern about the have been given to them and there has been much finding of nitrosodi-n-butylamineŽ. NDBA in some less interest in non-volatile nitrosamines or in ni- meat products, albeit at low concentrationswx 50 . This trosamides, although the latter are by no means to be unusual nitrosamine, which induces tumours of the ignored. Some non-volatile nitrosamines have been urinary bladder in rats and other specieswx 9 , was studied and are of little interest because they appear traced to rubber netting used to tie up the cured meat to be non-carcinogenic, including nitrosoproline, ni- after processing; derivatives of dibutylamine are used trosohydroxyproline, nitrosopipecolic acid, nitrosoth- in rubber manufacture and apparently become ni- ioproline and nitroso-4-methyl-thioprolinewx 27,28 ; trosated at some stage and the NDBA then migrates they have been detected and quantified in cured into the meat. Any time food comes into contact with meats, but their main toxicological interest lies in a rubber product, it can be expected that ni- their ability to act as nitrosating agents, for transni- trosamines in the rubber will migrateŽ. slowly into trosationwx 29 , and they can be thermally decarboxyl- the food. The process of manufacture of rubber atedwx 14 to produce cyclic nitrosamines which are invariably results in the formation of nitrosamines as potent carcinogens. by-products, which can attain considerable concen- Nitrosopyrrolidine was discovered in the vapours trationsŽ. many parts per million or more . Another from fried bacon and also in fried bacon itself, unusual nitrosamine, nitrosodiphenylamine, has been mainly in the fat, at concentrations approximating reported in applesŽ which had been treated with 100 ppb, which is considered quite significant; 100 g diphenylamine. . The concentration of the ni- of such fried bacon would contain 10 mg of nitrosamine was 2 to 6 mgkgy1, there being a higher trosopyrrolidine, which is one of the more potent concentration in the skinswx 33 . This is probably of carcinogenic nitrosamines. The nitrosopyrrolidine minor importance, because of the weakness of ni- might have arisen, at least in part, by decarboxyla- trosodiphenylamine as a carcinogen, although it does tion of nitrosoproline, or by some other mechanism; cause bladder tumours in ratswx 34 . as suggested by Loeppky et al.wx 30 ; there was also The pattern of nitrosamine content was similar in NDMA in cooked bacon, although at lower levels. nitrite or nitrate-treated products throughout the W. LijinskyrMutation Research 443() 1999 129±138 133 world, although in Japan and East Asia, where rela- once thought to be a source of methylnitrosourea tively little meat is eaten, the focus has been on fish, after nitrosationŽ and therefore likely to be present in nitrite-treated, smoked or otherwise processed. There cured meats. but there has been no good evidence have been persistent reports, e.g., from Hong Kong that the yield of MNU from a reaction of creatinine wx35 , of the relation between the high incidence of with nitrite is substantial. A similar alkylnitrosamide, nasopharyngeal cancer and the consumption of ni- methylnitrosourethaneŽ methylnitrosocarbamic acid trite-containing smoked fish as a major component ethyl ester. , has induced tumours of the nervous of theŽ. Chinese diet. NDMA has been identified in system transplacentally in ratswx 37 . Since many samples of the fishŽ reminiscent of Ender and Ceh's widely used pesticides are esters of N-methyl- story of the Norwegian fish meal. and, although carbamic acid and are very easily nitrosatedwx 38 , it is NDMA is considered mainly an inducer of liver feasible that residues of their N-nitroso derivatives tumors and lung or kidney tumors in rats, there have could be ingestedŽ. or formed in vivo and induce been experiments in which it has induced tumors of nervous system tumours in children. However, there the nasal cavity in rodents, albeit these are morpho- seem to have been no transplacental studies in rats or logically different from human nasopharyngeal tu- other animals of any such nitrosated pesticide. mors. Because of the well-known differences be- Another possible directly acting N-nitrosamide tween species in response to N-nitroso compounds that might be present in meatŽ and thereby related to wx8 , particularly in target organ, it is unwise to as- tumours of the nervous system. is methylni- sume that a particular N-nitroso compound would trosoguanidine, because guanidine is a constituent of induce the same tumors in humans as in any of the meat. Its derivative, methylnitrosonitroguanidine common experimental animals. Ž.MNNGÐan artificial compound , is one of the It was discovered a long time ago that alkylni- most common compounds in carcinogenesis and mu- trosoureas are potent inducers of tumours of the tagenesis studies. Alas, methylnitrosoguanidine is a brain and nervous system in rats and miceŽ but not in very unstable compound and has yet to be isolated, hamsters, gerbils or Guinea pigs, Kleihues et al.wx 36 . although evidence has been obtained for its presence especially when administered transplacentally via in solution. Little is known of its toxic, mutagenic or pregnant animals in the latter stages of pregnancy. carcinogenic propertiesŽ it could be formed tran- This has led to the search for a source of alkylni- siently in solution. and feeding tests of methylguani- trosoureas in the human diet as an explanation of dine and nitrite in rats to provide evidence of its brain tumours in humans, especially children; there formation in the stomach failed to give rise to any are few known nervous system carcinogens and none tumours. So evidence that any particular N-nitroso as effective as alkylnitrosoureas. There have been a compoundŽ. or, indeed, any other known carcinogen number of epidemiological studies of cured meat in the diet is responsible for brain tumours in chil- consumption by pregnant women who bore children drenŽ. or in adults is bleak, and research activity in that later developed tumours of the nervous system this area is rare. and none have been conclusive, although some were Although for many years there was slow progress suggestive. In particular, there seems to be a relation in understanding the magnitude of the problem of between the consumption of `hot dogs'Ž Frankfurter nitrosamines in food, it could not match the intensity sausages. in the USA and pediatric nervous system of effort following the discovery of NMDA in beer, tumors, the incidence of which is significantly ele- as a result of a comprehensive survey of ni- vated in children who consume five or more sausages trosamines in food and beverageswx 39,40 . No doubt a weekwx 18 . beer contaminationŽ. Table 1 was considered a more In spite of much searching, there has been little or serious matter than other foods regarding contamina- no evidence of the presence of methylnitrosourea or tion with carcinogens, especially in Germany, so the any other alkylnitrosourea in cured meats, although problem was solved with some haste. The highest Mende et al.wx 25 have inferred the presence of such concentrations of NDMA were found in `Rauchbier' compounds because of the methylating capacity of Ž.dark or smoky beer , high in malt content, so the the material. CreatinineŽ. a component of meat was origin of the NDMA was traced to alkaloids, horde- 134 W. LijinskyrMutation Research 443() 1999 129±138

Table 1 of NDMA and 100 ppb of nitrosopyrrolidine would NitrosaminesŽ. NDMA in beer consume 1 mg of NDMA and 10 mg of NO-Pyrr. Country Type of beer Maximum Intake of nitrosamines from other foods are simi- y1 concentration Ž.mgl lar, but might be repeated daily among people who Austria Lager 10 are fond of cured meats. Japanese people and other Belgium Strong lager 12 Eastern peoples often eat fish treated with nitrite Canada Beer 5 r Czechoslovakia Lager 2.5 and or smoked, in which the levels of nitrosamines Denmark Lager 0.5 are often somewhat higher than in Western cured France Lager 7 meats, and the intake of nitrosamines in these re- Germany Lager 1 gions might be more than traces. In the case of Hong Rauchbier 68 Kong and other coastal Chinese cities, it is tempting Sweden Lager 1 Switzerland Strong lager 9 to believe that the high incidence of nasopharyngeal United Kingdom Strong pale ale 8 cancer in these societies is due to consumption of USA Beer 8 NDMAŽ. and possibly other nitrosamines in smoked fish, which is eaten in large quantities; in some experiments in rats and hamsters, NDMA has in- nine and gramineŽ. and perhaps others present in duced tumours of the nasal cavity. The nitrosopiperi- barley and hence, in the malt. These alkaloids were dine in some samples of cured meatsŽ up to 50 or derivatives of dimethylamine and were easily ni- 250 ppb. almost certainly arises from nitrosation of trosated, in this case, by nitrogen oxides as the piperineŽ. and other piperidine derivatives in the nitrosating agentwx 41 . The nitrogen oxides were in spices used to flavour the meats. These concentra- the flue gases from the burning of the fuel used to tions of nitrosamines in selected cured meats provide heat the malt and allowed to come in contact with the consumers substantial exposure to these potent the latter. The solution to the problem was to se- carcinogens. quester the malt from the gases, which was the In Table 2 is shown the types of food commonly reason some beers seemed to be free of NDMAŽ as containing nitrosamines and the highest concentra- was proclaimed in their advertising. . Since that time, tion reported of each nitrosamine. This list is not beers have had a greatly reduced nitrosamine con- comprehensive and many of the references in this tent; before that time, beer in Germany and several review catalogue extensive results from analytical other countries was a major source of exposure of surveys of dietswx 8,27,28,42 . The diets of many people to carcinogenic nitrosamines, because of the countries and societies vary in their content of ni- very large quantities of beer that were drunk. One trosamines, many are low and others are relatively liter of beer containing 50 ppb of NDMAŽ by no high, depending on the types of food which contain means the highest concentration found. , for example, nitrosamines. Food has been surveyed for ni- provided 50 mg to the drinker, who might drink trosamines in only selected countries, including several liters a day. NDMA was also present in Kashmir, Thailand, China, Japan, as well as the whiskies of various kinds, no doubt arising from the United States, Canada and countries of Europe. The same sourceŽ. barley malt , but at lower concentra- amounts consumed by individuals vary widely, so tions than in beer and posing a lower cancer risk, that average consumption has little meaning in rela- because whisky is consumed by most people in tion to carcinogenic risk of individuals. For example, smaller amounts than beer. Although changes in beer there are peopleŽ especially children, who might be manufacture, and increased regulation, have reduced particularly susceptible to carcinogens. who eat ba- the level of nitrosamines in beer to 5 ppbŽ which is con, ham or sausages daily and there are others generally `accepted'. , it must be remembered that 1 l Ž.including this author and vegetarians who consume of beer containing 5 ppb of NDMA provides 5 mgof none, and everything in between. The consumption NDMA to the drinker, who might well drink several of nitrosamines by those who eat a lot of cured liters in a day. On the other hand, someone who eats meats will almost certainly be much greater than by 100 g of baconŽ. a good serving containing 10 ppb vegetariansŽ unless the latter drink vast amounts of W. LijinskyrMutation Research 443() 1999 129±138 135

Table 2 Nitrosamines found in foods Nitrosamine Food and highest concentration recorded Ž.mg kgy1 Relative carcinogenicity N-Nitroso- Dimethylamine beerŽ. 8 , bacon Ž 17 . , cheese Ž. 5 , cured meats Ž 22 . , qqqq sausageŽ. 12 , Thai fish Ž. 25 , smoked pickled fish Ž. 32 , dried milk Ž 4.5 . , broiled squid JapanŽ. 300 , salted meat Russia Ž. 54 , etc. Diethylamine corn bread, seafood ChinaŽ. 4.8 , sausages Ž. 10 , cheese Ž. 20 qqqq Pyrrolidine fried baconŽ. 100 , sausages, China, Germany Ž. 45 , qqq broiled squid JapanŽ. 10 , smoked meat Ž. 10 , ham Ž. 36 Piperidine bologna, sausagesŽ. 50 , spiced smoked meat Ž. 9 , qqq Chinese picklesŽ. 14 , Thai fish Ž. 23 , Thai pork Ž. 6 , Tunis stew base Ž. 43 Methylbenzylamine corn bread, China Ž.)100 qqqq Thiazolidine smoked porkŽ. 5 , sausage Ž. 5 , smoked oysters Ž 109 . , ± fried baconŽ. 240 , cured meats Ž. 27 , smoked fish Ž. 2 Proline fried baconŽ. 68 , cured meats Ž 400 . , smoked pork Ž 2100 . , ± sausageŽ. 940 , smoked oyster Ž. 167 Thiazolidine- fried baconŽ. 14000 , cured meats Ž. 3900 ± Carboxylic acid smoked fishŽ. 1600 ± beer. . I heard it said at one time that one quarter of been few reports of the occurrence of alkylni- the meat consumed in the USA is cured with nitrite, trosamides, especially alkylnitrosoureas, in foods. In illustrating the magnitude of the problem, even rats, methylnitrosourea and ethylnitrosoureaŽ and though the amount of nitrite used in meat curing is higher homologues. given transplacentally to preg- now more firmly controlled than one or two decades nant females have induced tumours of the nervous ago. There have been estimates published of the systemŽ. including brain in a high proportion of the average intake of nitrosamines by people in a partic- offspringwx 44 . The doses have often been quite small ular country or region and these have been of the to produce this effect. order of 1 mg persony1 dayy1. However, as dis- One reason for the failure to detect alkylni- cussed above, this value can be close to zero in many trosoureas in food is probably their instability, espe- people and considerably greater in others who con- cially in alkaline solution. Mende et al.wx 25 have sume a lot of beer or cured meats. inferred the presence of methylnitrosourea in some The main carcinogenic N-nitroso compounds foods by an indirect method involving the measure- found in food throughout the world are NDMA, ment of methylation of nitroso-t-butylglycine by di- NDEA, nitrosopyrrolidine and nitrosopiperidine, with azomethane released from the directly acting nitrosodi-n-butylamine occasionallyŽ derived from nitroso-alkylurea. the food's contact with rubber. . This is reflected in surveys of foods from a variety of countries. How- ever, there have been reportsŽ. not always verified of 4. Quantitative considerations some unusual nitrosamines in some foods from China, Japan and other oriental countries. These The concentrations of N-nitroso compounds in nitrosamines include nitroso-1-methyl-acetonyl-3- foods are often tiny, and human exposure to them methylbutylamine, methylnitrosobenzylamine and ni- from this source is quite small, but the consequences trosodibenzylaminewx 21 . Most `food nitrosamines' to people are not to be ignored for two reasons. gave rise to tumours of the liver or esophagus when Firstly, because of the great carcinogenic potency of fed to rats, but would induce different tumors in this group of carcinogensŽ. in animal studies , particu- hamsters, which have never been reported to bear larly some of the simpler ones and, secondly, be- tumours in the esophagus. cause nitrosamines might well be more effective In spite of much searching, particularly in connec- Ž.dose-for-dose in humans than they are in experi- tion with pediatric brain tumourswx 43 , there have mental rodents, as they are more effective in rats 136 W. LijinskyrMutation Research 443() 1999 129±138 than they are in mice or hamsters. Evidence that this large amount of cured meats eaten by their mothers is so lies in a comparison of the dose of nitrosamines while they were pregnant. The epidemiological stud- to a heavy cigarette smokerwx 42 , 20 cigarettes dayy1 ies are complicated by the formation of N-nitroso or approximately 4 mg nitrosamines or 0.06 mg compounds from ingested secondary and tertiary dayy1 kgy1 body weightÐconsisting largely of the amines while nitriteŽ from food or formed endoge- rather weak carcinogen, nitrosonornicotine, and the nouslywx 47 and present in saliva. is in the stomach almost inactive nitrosoanatabine, together with ap- wx14 . This, of course, adds to the total exposure to proximately 20%Ž corresponding to 0.012 mgkgy1 carcinogenic N-nitroso compounds. dayy1 .Ž of the potent carcinogen NNK 4-methyl- An exception to this failure to find an epidemio- nitrosamino-3-pyridylbutanone. . Hoffmann and logical correlation appears to be the high incidence Hechtwx 45 assume that the nitrosamines play a major of esophageal cancer in certain parts of China and in role in the increased cancer risk of tobacco chewers other parts of the world, where nitrosamines have and tobacco smokers; this dose is responsible for a been found in substantial concentrations in native 10% incidence of cancer in heavy smokers. In com- foodswx 48 . parison, in the large scale dose-response study of the more potent NDMA and NDEA in male rats carried out two decades ago, the minimal effective dose 5. The future control of nitrites Ž.causing a 1±4% tumor incidence was 40 mg dayy1 kgy1 body weightwx 46 . Even considering that expo- The focal point of attempts to reduce the concen- sure of the rats lasted 2±3 years, while exposure of tration of N-nitroso compounds in foods is the con- humans could last 40±50 years, the difference of trol of nitritesŽ. or other nitrosating agents which can more than three orders of magnitude in nitrosamine come into contact with nitrosatable amino com- exposure between the experimental animals and pounds. There has been notable success in reducing smokers indicates that humans are more responsive the concentration of NDMA in beer by controlling to carcinogenic nitrosamines than are rats. the exposure of malt to nitrogen oxides, effectively It has been difficult, so far, to relate ingestion of stopping nitrosation of the alkaloids in the malt. food containing N-nitroso compounds with a particu- There has also been some success in reducing the lar incidence of cancer. The finding that certain amount of nitrite used in meat curing, which is the foods contained carcinogenic nitrosaminesŽ albeit at source of most of the N-nitroso compounds in cured low concentrations. led to the suspicion that they meats. Since the rate of formation of nitrosamines is might be responsible for the high incidence of stom- proportional to the square of the nitrite concentra- ach cancer in many parts of the world and in some tion, reduction of the latter has an enlarged effect in societies among them. It was a natural assumption reducing the amount of nitrosamine formed. The that direct contact with these carcinogens in the reduction in nitrite and the virtual elimination of stomach was responsible. However, good evidence nitrate has been both by voluntary action by manu- has been lacking connecting the exposure with the facturers and, in many countries, by government cancer. This is understandable because nitrosamines regulation. Among the regulations has been the re- are prime examples of systemic carcinogens that do duction of nitrite permitted in bacon from 200 to 125 not act locally and very few of those tested in ppm and the elimination of nitrite from certain types animals have induced tumours of the glandular stom- of sausageŽ. in Norway , as well as a reduction in the ach, such as humans possess; some nitrosamines do use of nitrite in fish. These controls have led, over induce tumours of the squamous or non-glandular the past 20 years, not only to a reduction in the stomach which is present in some rodents. nitrosamine content of many common cured meats, Correlation of other types of cancer with con- but also to a lessened exposure to nitrite which sumption of foods containing N-nitroso compounds participates in endogenous formation of N-nitroso has not been very successful, although there have compounds. From the early 1970s, there was a grad- been several epidemiological investigations of a con- ual reduction of the amount of nitrosopyrrolidine in nection between brain tumours in children and the fried bacon from above 100 ppb to approximately 30 W. LijinskyrMutation Research 443() 1999 129±138 137 ppb or less in the late 1970s, but more recently, there wx13 W. Lijinsky, In vivo testing for carcinogenicity, in: P.L. has been an increase to as much as 65 ppb of Grover, C.S. CooperŽ. Eds. , Chemical Carcinogenesis and Mutagenesis, Springer, Berlin, 1990, pp. 179±209. NO-PYR, up to 110 ppb of all volatile nitrosamines wx wx 14 W. Lijinsky, S.S. Epstein, Nitrosamines as environmental in fried bacon and 85 ppb in the fried-out fat 49 . carcinogens, NatureŽ.Ž. London 225 1970 21±23. There is less evidence that there is action to reduce wx15 J. Sander, F. Schweinsberg, Untersuchungen uberÈ die Enste- human exposure to N-nitroso compounds in fish, hung von Nitrosaminen und Nitrosamiden im Menschen, im smoked or nitrite-cured, in those countries in which Tier und in Nahrungsmitteln, Zbl. Bakt. Hyg. I Abt. Orig. B Ž. fish is a large part of the dietŽ Japan, China, Russia, 156 1972 321±340. wx16 J. Sander, G. Burkle,È Induktion maligner Tumoren bei Ratten etc.. . durch gleichzeitige VerfutterungÈ von Nitrit und sekundaren However, it is to be hoped that such action will be Aminen, Z. Krebsforsch. 73Ž. 1969 54±66. taken voluntarily, since governments seem to have wx17 W. Lijinsky, H.W. Taylor, Nitrosamines and their precursors been rather immobile on this issue, although infor- in food, Cold Spring Harbor Symposium on the Origins of mation about the possible carcinogenic risk has been Human Cancer, Book C, 1977, pp. 1579±1590. wx18 S. Preston-Martin, J.M. Pogoda, B.A. Mueller, E.A. Holly, available for decades. This action will surely lower W. Lijinsky, R.L. Davis, Maternal consumption of cured exposure of human populations to the broadly acting meats and vitamins in relation to pediatric brain tumors, carcinogenic N-nitroso compounds and thereby re- Cancer Epidemiol., Biomarkers Prev. 5Ž. 1996 599±605. wx duce the risk of cancer to these people. 19 D.H. Fine, F. Rufeh, Description of the thermal energy analyzer for N-nitroso compounds, IARC Scientific Publica- tions No. 9Ž. 1974 40±44. wx References 20 C.L. Walters, Group analysis of N-nitroso compounds, Das Nitrosamin-Problem, Verlag Chemie, Weinheim, 1983, pp. wx1 R. Doll, R. Peto, The causes of cancer: quantitative estimates 93±96. of avoidable risks of cancer in the United States today, J. wx21 S. Lu, M. Li, J. Chuan, M. Wang, Y. Wang, A new Natl. Cancer Inst. 66Ž. 1981 1191±1308. N-nitroso compound, N-3-methylbutyl-N-1-methylacetonyl- wx2 R. Langenbach, S. Nesnow, J.M. Rice, Plenum, New York, nitrosamine, in corn bread inoculated with fungi, Scientia pp. 509±529. Sinica 22Ž. 1979 601±607. wx3 N. Koppang, An outbreak of toxic liver injury in ruminants, wx22 G.M. Singer, C. Ji, J. Roman, M.-H. Li, W. Lijinsky, Ni- Nord. Vet. Med. 16Ž. 1964 305±322. trosamines and nitrosamine precursors in foods from Linxian, wx4 F. Ender, G. Favre, A. Helgebostad, N. Koppang, R. Mad- China, a high incidence area for esophageal cancer, Carcino- sen, L. Ceh, Isolation and identification of a hepatotoxic genesis 7Ž. 1986 733±736. factor in herring meal produced from sodium nitrite pre- wx23 W. Lijinsky, R.K. Elespuru, A.W. Andrews, Relative muta- served herring, Naturwissenschaften 51Ž. 1964 637±638. genic and prophage-inducing effects of mono- and dialkyl- wx5 L.K. Keefer, P.P. Roller, N-Nitrosation by nitrite ion in nitrosoureas, Mutat. Res. 178Ž. 1987 157±165. neutral and basic medium, Science 181Ž. 1973 1245±1247. wx24 S. Ivankovic, F. Klimpel, M. Wiessler, R. Preussmann, Car- wx6 D. Hoffmann, K.D. Brunnemann, J.D. Adams, S.S. Hecht, cinogenicity in BD-IX rats of 7 homologues of N-nitroso-N- Formation and analysis Of N-nitrosamines in tobacco prod- n-alkylureas in different stages of postnatal development, ucts and their endogenous formation in consumers, IARC Arch. Geschwulstforsch. 51Ž. 1981 187±203. Scientific Publications No. 57Ž. 1984 743±762. wx25 P. Mende, B. Spiegelhalder, R. Preussmann, A sensitive wx7 P.N. Magee, J.M. Barnes, The production of malignant pri- analytical procedure for detection of N-nitrosamides via their mary hepatic tumours in the rat by feeding dimethylni- alkylating activity, Food Chem. Toxicol. 27Ž. 1989 475±478. trosamine, Br. J. Cancer 10Ž. 1956 114±122. wx26 L. Hedler, P. Marquardt, C. Schurr, The relation between the wx8 W. Lijinsky, Chemistry and Biology of N-nitroso Com- determinable quantities of volatile N-nitroso compounds and pounds, Cambridge Univ. Press, Cambridge, UK, 1992. the peroxide number in soya bean oil, IARC Scientific wx9 R. Preussmann, B.W. Stewart, N-Nitroso carcinogens, in: Publications No. 14Ž. 1976 361±374. C.E. SearleŽ. Ed. , Chemical Carcinogens, American Chemi- wx27 J. Gao, J.H. Hotchkiss, J. Chen, Regional differences in cal Society Monograph No. 182, 1984, pp. 643±828. N-nitrosamine content of traditional Chinese foods, IARC wx10 P. Bogovski, S. Bogovski, Animal species in which N-nitroso Scientific Publications No. 105Ž. 1991 219±222. compounds induce cancer, Int. J. Cancer 27Ž. 1981 471±474. wx28 N.P. Sen, S.W. Seaman, P.A. Baddoo, N-Nitrosothiazolidine wx11 W. Lijinsky, R.M. Kovatch, Carcinogenesis by nitrosamines and nonvolatile N-nitroso compounds in foods, Food Tech- and azoxyalkanes by different routes of administration to nol. 39Ž. 1985 84±88. rats, Biomed. Env. Sci. 2Ž. 1989 154±159. wx29 S.S. Singer, W. Lijinsky, G.M. Singer, Transnitrosation: an wx12 W. Lijinsky, Lifespan and cancer: the induction time of important aspect of the chemistry of aliphatic nitrosamines, tumors in diverse animal species treated with nitrosodieth- IARC Scientific Publications No. 19Ž. 1978 175±178. ylamine, Carcinogenesis 14Ž. 1993 2373±2375. wx30 R.N. Loeppky, W. Tomasik, J.R. Outram, A. Feicht, Ni- 138 W. LijinskyrMutation Research 443() 1999 129±138

trosamine formation from ternary nitrogen compounds, IARC of volatile nitrosamines in food: a survey of the West Ger- Scientific Publications No. 41Ž. 1982 41±56. man market, Oncology 37Ž. 1980 211±216. wx31 H.S. Rosenkranz, G. Klopman, Computer-automated struc- wx41 M. Mangino, R.A. Scanlan, T.J. O'Brien, N-Nitrosamines in ture evaluation of the carcinogenicity of N-nitrosothiazoli- beer, in: R.A. Scanlan, S.R. TannenbaumŽ. Eds. , N-Nitroso dine and N-nitrosothiazolidine-4-carboxylic acid, Food Compounds, American Chemical Society Symposium Series Chem. Toxicol. 25Ž. 1987 253±256. No. 174, 1981, pp. 229±245. wx32 W. Lijinsky, L.K. Keefer, J.E. Saavedra, T.H. Hansen, R.M. wx42 A.R. Tricker, N-Nitroso compounds and man: sources of Kovatch, W.E. Middler, A.T. Miller, Carcinogenesis in rats exposure, endogenous formation and occurrence in body by cyclic N-nitrosamines containing sulfur, Food Chem. fluids, Eur. J. Cancer Prev. 6Ž. 1997 226±268. Toxicol. 26Ž. 1988 3±7. wx43 S. Preston-Martin, B.E. Henderson, N-Nitroso compounds wx33 T.J. Lillard, J.H. Hotchkiss, N-Nitrosodiphenylamine in and human intracranial tumours, IARC Scientific Publica- diphenylamine-treated apples, in: R.N. Loeppky, C.J. tions No. 57Ž. 1984 887±894. MichejdaŽ. Eds. , Nitrosamines and Related N-nitroso Com- wx44 S. Ivankovic, Teratogenic and carcinogenic effects of some pounds: Chemistry and Biology, American Chemical Society chemicals during prenatal life in rats, Syrian golden hamsters Symposium Series No. 553, 1994, pp. 358±360. and minipigs, Perinatal Carcinogenesis, Natl. Cancer Inst. wx34 R.H. Cardy, W. Lijinsky, P. Hildebrandt, Neoplastic and Monograph No. 51Ž. 1979 103±115. non-neoplastic urinary bladder lesions induced in Fischer 344 wx45 D. Hoffmann, S.S. Hecht, Nicotine-derived N-nitrosamines rats and B6C3F1 hybrid mice by N-nitrosodiphenylamine, and tobacco-related cancer: current status and future direc- Ecotoxicol. Environ. Safety 3Ž. 1979 29±35. tions, Cancer Res. 45Ž. 1985 935±944. wx35 J.H.C. Ho, Some epidemiologic observations on cancer in wx46 R. Peto, R. Gray, P. Brantom, P. Grasso, Dose and time Hong Kong, Natl. Cancer Inst. Monograph No. 53Ž. 1979 relationships for tumor induction in the liver and esophagus 35±47. of 4080 inbred rats by chronic ingestion of N-nitroso- wx36 P. Kleihues, R.M. Hodgson, C. Veit, F. Schweinsberg, M. diethylamine or N-nitrosodimethylamine, Cancer Res. 51 Wiessler, DNA modification and repair in vivo: towards a Ž.1991 6452±6469. biochemical basis of organ-specific carcinogenesis by meth- wx47 S.R. Tannenbaum, D. Fett, V.R. Young, P.D. Land, W.R. ylating agents, Organ and Species Specificity in Chemical Bruce, Nitrite and nitrate are formed by endogenous synthe- CarcinogenesisŽ. 1983 , Plenum, New York, 509±529. sis in the human intestine, Science 200Ž. 1978 1487±1489. wx37 T. Tanaka, Transplacental induction of tumours and malfor- wx48 C.S. Yang, Research on esophageal cancer in China: a mations in rats treated with some chemical carcinogens, review, Cancer Res. 40Ž. 1980 2633±2644. IARC Scientific Publications No. 4Ž. 1973 100±111. wx49 B.J. Canas, D.C. Havery, F.L. Joe, T. Fazio, Current trends wx38 R.K. Elespuru, W. Lijinsky, The formation of carcinogenic in levels of volatile N-nitrosamines in fried bacon and fried- N-nitroso compounds from nitrite and some types of agricul- out bacon fat, J. Assoc. Off. Anal. Chem. 69Ž. 1986 1020± tural chemicals, Food Cosmet. Toxicol. 11Ž. 1973 807±817. 1021. wx39 B. Spiegelhalder, G. Eisenbrand, R. Preussmann, Occurrence wx50 N.P. Sen, Recent studies in Canada on the occurrence and of volatile nitrosamines in food: a survey of the West Ger- formation of N-nitroso compounds in foods and food contact man market, IARC Scientific Publications No. 31Ž. 1980 materials, IARC Scientific Publications No. 105Ž. 1991 232± 467±479. 234. wx40 B. Spiegelhalder, G. Eisenbrand, R. Preussmann, Occurrence