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I J i I 432 Nitrosamines : ubiquitous carcinogens? New Scientist 23 August 19: Known to occur naturally in human food, nitrosamines are also manufactured by simple chemical reactions :- •I,^ e•#%1++arh. These chemicals have been shown to be extremely carcinopenic at very low doses in experimental animals. What, therefore, is tne na:aru wo sa,oo.: Peter Magee is professor of experimental biocbemistry at the Courtauld Institute of' Biochemistry. London In the mid 1950s I was working with Dr John °---^- -• *s.o tutpettrat Research Council's Toxicology Research Unit at Carshatton tn Surrey. We were interested in the acute toxi- :;cy of dimethylnitrosamine on the livers of a number of experimental animals. In 1956 we discovered that at very low dosage levels ~(50 part per million of diet) dimetbylnitrosa- mink was carcinogenic-it induced liver tam• ours between the 29tb and 40th week of treatment in almost all the animals (rats) given it. This was the first indication that nitrasamines (see Figure 1) could cause !°?l~eer. Now there are more than 100 known oatciaogenic nitrosamines. Thg occurrence of low levels of these compounds in the human environment is currently causing a great deal of ¢oncern about possible health hazards. It is interestipg, in the light of our find- ings in the 1950s. that as long ago as 1937 Hugo Freund of Detroit gave a detailed dini- eal and pathological report of two cases of acute dimethylnitrosaarine pois,oning in lab• oratory workers. In his paper, entitled °Clini- eal manifestations and studies of parenchy- matous hepatitis", he gave no indication that the damage was concerned with nitrosamine toxicity. After our reports of the toxicity and o~.a.ar• einogenicity of the cbemical, the position dI nged. Many people followed up our work, notably Professor Hermann Druckrey and his colleagues at the University of Freiburg. Dr}lckrey embarked on a systematic study of the relationship between chemical structure and carcinogenic action of a range of N• nitroso compounds. By the late 1960s ft had become apparent that many nitrosamittes could induce cancer in rats and other experi- mental animals. But more interesting, ft tnrned out that nitroso compounds with diffe- reitt structures caused tumours in different organs. Notable examples of this organ selectivity, or organotropism as Druckrey has called it, are the production of bladder tumours by dibutylnitrosamine (Figure 1) and the ten• dency for unsymmetrical nitrosamines, for example N•nitrosomethylaniline (Figure 1), to induce tumours of the oesopbagus. Perhaps the most interesting finding was that N•nitro• sahmethylurea, after repeated intravenous ad- ministration, produces large numbers of ttxmours of the brain and other parts of the nervous system. This was the first eSective ezperimental method for brain tumour induc- tion other than by direct application of a chemical carcinogen to the brain and has been used extensively by experimetttal neuro- plathologists. N•nitrosoethylurea has the remarkable ppacity to induce nervous system tumours in the offspring of the mother rats that have received it during the latter part of pregnancy. tJruckrey and his , colleagues showed th brain tumours were induced in some of t1 p~Y~oyr ..:.c.. :. hprl r ceived as little as one fiftieth of the medi: lethal dost of nitrosoethylurea. As such 9Mse bas no detectable toxic effect on tl mother animals the implications of this fi:n Jag are obvious. It is also notable that tl experitneqtal.demonstration of transplaceni tarcinogenesis occurred before the appe: ance of thk recent cUnitssl evidence suggesti transplaccntal induetion of vaginal canc in yotmg women whose mothers had be treated with diethylstilboestrol duri lregnalmy• . . The induction of tumouri by single doa of nitrosd compounds is interesting, becaL it has also been observed after treatment newborn and adult animals, and because I nit.osamines (particularly the amide dert tives) may be very rapidly eliminated ft tht body. It seems that all the animal spec tested so far-including monkeys and ot] memmals, birds, 'amphibia and fish-,• ansceptible to tumour induction by uitra mines. This implies that man is probably a at risk. Maqy N-nitroso eompounds are equally potent mutagens as carcinogens. The che cally utuKable amide derivatives, nota N nitroso-N%nitroo-N methylguanidine (Fig 1), are very effective mutagens in atl usual mic"bial test systems, but the m chemieal$+ stable nitrosamines are not. I is esplained by the widely accepted view t the nitroso compounds are not, themseh biologically active, but produce their eHi via a chemically reactive intermediate. 7 intermediate may be fonmed with or witt the action of enzymes. The chemically stable nitroaamines dec Pon sbo mat~)ism~ oocarrtng mainly bnt n~e:x aively ia the liver. The esnymes eottcer have features in common with the weli krt groups of enzymes (mitsnsomal hydroiWla that are responsible for the metabolisa most ampounds toreiga to the boftr. general these reactions reduce the toxieit the compo»nd and are thus beneficial. sometimes, as with the nitrosamines, the .arse is the case, and the decomposition duels.re more toxic and/or cardnogenic 1 the parent compounds. There is some evidence suggesting thal aites of tumour Induction by the nitrosaa (organotrophy) may be determined in by the presence of the aestasary emMt tbe target organs. Factors other than enz •ctivation must also be involved, bovv4 because the chemically unstable nitroao ctnogens can also show considerable o epsciRcitiet. Met4bolic sct'w*tL- '` • -eompounds aeeigi toN to b 18 0 8 ~

NeW Sc,entist 23 August 1973 concept that the chemical to which the animal is kacptlsed is often not the moleculat species which induces the tumour has given rise to a new group of terms: precarcinogen, for the parent compound; proximate carcinogen for more carcinogenically active decomposi- tion oroducts: and ultimate carcinogen for tne product wnrcn reacry wun souIc uu,ioI cellular component and thus Induces cancer. The well known American husband and wife team, James and Elizabeth Miller, point out that ultimate carcinogens are ouen eleetrophilic (positively charged) Feactants which combine with aucleophilic (negatively charged) centres in nucleic acids, roteins, and other cell components. Severa~nitroso carcinogens are known to give rise to alkylat- ing agents, possibly carbonium ions (which are positively charged), in the body. These may be the corresponding ultimate carcino- gens. It is also possible, however, that other eareinogenic decomposition products may be involved. Although there is a large amount of information on the tiaetabo)ism mf nitro- samines and other carcinogens, the way in which the active metabolites transform normal cells into cancer cells is still almost entirely unknown and is being actively . studied in laboratories throughout the world. The nitrosamines have many advantages for use in such studies, particularly in work on whole animals. Interest in •and concern about the possible cancer hazards of nitrosatnines for man arose from observations of an outbreak of a severe 1 43: and occasionally fatal disease of sheep nt Norway in the. harly 1960s. A number of t}ai sheep become fll with no immediately ap~pe rent cause. Subsequently they wer'e found tc have severe liver injury. Investigations , re vealed that an the aifected sheep had eater partially decomposed fisbmeal containing hit s.LC .a a p,w....~..... . _ • _.,, _,- considerable quantities of amittes, includini dimetbylamine and trimethyla>Atine, the pos sibility that nitrosamines might have beer ~ Iurweu ww wuaeuc.u: w...: a...:::. ::.. L _- --' rect. Chemical analysis dearly demonstrated the presence of dimethylnitrosamiae in t1qE meal in amounts sufiicient to explain the li'ver damage in the sheep. This dramatic demonsmtion of the fortna tion of toxic amounts of a powerfully carano genic nitrosamine in food for sheep led tc concern that a similar sir--tiioti might ockur in food for human consumption for whict nitrite is a permitted additive. In the UK such foods include various cured meats sdtcl as bacon and ham and certain dbeeses. Exteit sive programmes of food analysis were sel in train throughout the world and it w a: immediately apparent that the availabh microanalytical methods for nitrosamine: were quite inadeqnate. Reliable and sensitivE methods, involving gas-liquid chromatograph3 and mass spectrometry, hav~.- now been de veloped at the Laboratory of the Gooernmen, Chemist in London and in other laboratories We now know that very small amounts (in tbi microgram per kg range), of nitrosamines CH3 CH3 ~ CHSCH=Cri2 CH2 ` CH=-CH: ~MNO NNO / NNO / ~ ~NNO CHS ' CHsCHZCH2 CH2 CH -CH / CX = = N•oitrosodimethylamine N•nitrosodibutylamine N-nitrosopyrrolidine . N•nitr+osometbylaniline (Dimethylnitrosamine) (Dibutylnitrosamine) The chemistry of nitrosamines Nitrosamines are N•nitroso compounds that have been well known to organic chemists for many years. They are readily formed by the reaction of secondary CN3\NNO emines with nitrous acid (nitrite in an acid medium). Examples of some t N-nitroso compounds with important biological actions are shown above. There C=O are, of course, many more N-nitroso compounds, the prefix N being used to distinguish them from C-nitroso compounds in which the nitroso group is attached to a carbon atom. NNa The Figure shows examples of dialkyl. heterocylic, and arylalkylnitrosamines, N•oltnasometlty+lurea together with two N•nitrasoderivatives of simple amides, N•methylurea. and N'-nitro-N-guanidine. The amine derivatives differ from the nitrosated amides in their chemical stability. The former compounds are usually chemically atable 'in aqueous solution in the cold if kept in the dark, while the latter are unstable in alkali and may decompose more slowly at neutral pH. Nitroso derivatives of alkyl amides readily yield the corresponding diazoaikane on treatment with alkali and have been used extensively for alkylation reactions in synthetic organic chemistry. Exposure of N-nitroso compounds to ultraviolet light causes decomposition, with quantitative yield of nitrate (for which there are sensitive methods of ineasurement ). This photochemical decomposition is the basis of one of the methods for determining of nitroso compounds. , , N-aitnsso-N'attroNmetbyl=ttaddiae I• iZN 1800

1 I I t 434 Disorganisation of scientific research including dimethylnitrosamine and N-nitroso- pyrrolidine (Figure 1) may be present in bacon and other foods. At the moment the im- plication of these findings for human cancer induction are very difficult to assess. Anotner vck y n~t.r....., -, . _. _R.L .:..... samine problem has become clear from the work of Johannes Sander and others who have shown that, perhaps not surprisingly, carcinogenic nwvw...:.... .:..:= hc s^"^•I i^ the acid conditions of the mammalian stomach as a result of the simultaneous presence of appropriate amines and nitrites. The signifi- cance of this work is increased by the recent discovery that not only secondary but also tertiary amines can be nitrosated to yield car- t:inogenic nitrosamines. No one doubts the potentially adverse results of such endogenous nitrosamine formation because there have been numerous reports of the induction of tumours in experimental animals by the simultaneous administration of nitrites and various secondary or tertiary amines. The implications of the latter findings are particularly significant because many drugs and other environmental chemicals contain nitrosatable tertiary amino groups. These in- tragastric nitrosations can be prevented to varying extents by the simultaneous ingestion of ascorbic -acid .(vitamin C); this offers the possibility of reducing the carcinogenic hazard tNat might arise from them. The problem of hazard from ingestion of nitrites as food additives merges with that of nitrite intstke from other sources and with the general question of nitrates in the environ- tnent. Nitrites may occur in variable amounts New Scientist 23 August 19T." in spinach and some other vegetables, tivhere they are probabl) formed from nitrates bi the action of nitrate reductase e.nzymes. Thes+ enzymes are also present in the human ali mentary tract where, particularly in infants •t- -nv oroduce sufficient reduction of lhi nitrate in ItrinKing waka. ..• ...~~ . . acute nitrite poisoning with methaemotlobir semi:D. Nitrite is in the saliva of most humai subieds. again apparently as a result of re ducton of nitrate. it aecuu, ::....:.:;, :.L-: nitrites can get into the stomach from source other than food. Given that trace amounts of earcitiogeni nitrosam(nes occur in certain foods for buma consdmption. and that they can be forme in ti{e body, what are the implications ft human cancer risk? This question cannot t answered with any confidence in the preset state of knowledge of dose.response relatio ships in chemical carcinogenesis. Some peop believe that no dose of a carcinogen, bower small, can be regarded as completely saf this concept forms the basis of the weH-knov Delaney Cancer Clause of the United Stat Constitution. Others, however, feel that the may be defence mechanisms* available to s body sd,ch as Immunological survet7lattte incipient cancer cells, and DNA repair pi cessb:s which could imply that there arc dot of chemical carcinogens below which no r4 human hazard exists. At the moment the is a lot of research e:Hort, especially America, to clarify this situation by do response experiments involving very lat numbers of experimental animals (so call mega mouse experiments). "My thesis may be simply stated. It Is that In Britain we are spending a, great deal of money on research, that we have every reason to be disappointed by the productivity of that research, and that most attempts' to improve the situation have made It worse."-Professor Kenneth Metlanby in his presidential address to Section X of the British Association In Canterbl..y this week Professor Kenneth Melianby is director of the Nature Conservancy's Monks tti ood Experimental Station, Abbots Ripton The amount of money that we are spending on research, and the rate at which we have in- creased our spending v:ill be a surprise to many. The graph opposite shows how government grants to all the research coun• cils, and through the University Grants Com- mittee to the universities, have grown since the last war. The money provided has in- creased by a factor of nearly 400 for the research councils, and 200 for the univer- aities. Inflation has of course played a part, and the total government expenditure has in• creased some 20 fold in the same period, bu' nevertheless the share of this "cake" avail- able to the universities is 10 times, and to the research councils 20 times, greater than it was just before the 1989-45 war. Of the money so far discussed, all that voted to the research councils was clearly intended to support research. That for the universities is more difficult to apportion. In the pre-war period those who begrodged meagre Cd million the state gave to the versities and the high staff/student rat allowed to exist were told that about hali budget covered research, without which te Cyi g bf the right kind was impossible. Pa C*e research council money also wen Sversities who had additional sources ~ undations. 00 However, only about a sixth of the m '4"ioted by government for research today o the research councils, as Table i shows , The scientists supported by these fun most government laboratories form {ta the same scientific community and are su .Jo the same forces as those in uni'versitie '4eaeaech councils, and what I have •e ^fiere applies, in general, to them. Theit Cikets have increased much in the wme There has also been a timilar increa research spending by industry- I must ex