C&EN feature DR. JOHN H. WEISBURGER and DR. ELIZABETH K. WEISBURGER National Cancer Institute
CHEMICALS All t o o o f t e n , c h e m i s t s a r e n o t a w a r e of t h e cancer-inducing h a z a r d s of m a n y o f t h e c h e m i c a l s or classes of chemicals w i t h which t h e y work. Often, too, t h e h a z a r d is n o t k n o w n , a s in t h e c a s e o f n e w s t r u c t u r e s . B u t recognizing t h e risks, c h e m i s t s can take appropriate precautions t o p r e v e n t e x p o s u r e of p e r s o n n e l —and t o avoid exposure themselves —to carcinogenic agents whose e f f e c t s m a y n o t be d e t e c t e d until a f t e r f r e q u e n t exposure for many years
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Cancer research falls into two broad groupings—diagnosis and treatment, and etiology and prevention. Diagnosis and treatment relate to the methods leading to the discovery of a cancer already present, so that appropriate remedial measures such as surgery, irradiation, or chemotherapy can be used. Etiology and prevention deal with attempts to discover the causes and origins of neoplastic diseases, to understand the mechanism of their formation, and to delve into their inherent nature. The underlying idea for research on the etiology and prevention of cancer is that the disease can be prevented by modifying its course or by eliminating causative or accelerating factors. This report covers a portion of cancer research pertaining to etiology and prevention. In particular, the article discusses cancer induction by aromatic amines, azo dyes, nitrosamines, and mycotoxins. With these facts, chemists should be in a position to avoid the widespread use of chemicals that are suspect, and thus to circumvent cancer hazards to themselves, their communities, and their customers. Early Noted Hazards of Aromatic
Amines
In the middle of the 19th century, while searching for drugs, Perkin stumbled upon mauve, a dye, obtained by the oxidation of a mixture containing aniline. Further advances in the dye industry came about through the development of large-scale syntheses of aniline and derivatives, which served as raw materials for the budding industry. The first commercial dye establishments were founded between 1860 and 1880 in the Rhine Valley of Germany and Switzerland. In 1895, the German physician Rehn noted three cases of bladder cancer, not in a random population, but in employees of a factory making dye intermediates. Rehn attributed these cancers to his patients' occupation, from which evolved the label aniline cancer. Subsequent reports confirmed this relationship—there was usually a 15to 30-year interval between the time of original employment and the occurrence of bladder cancer—although the
As Causes Of
CANCER
label aniline cancer is now considered a misnomer. Animal tests showed pure aniline to be noncareinogenic. Attempts were made to demonstrate experimentally that some dye intermediates were carcinogenic. However, it was not until 1937 that Hueper, then at E. I. du Pont de Nemours and Co., and recently retired from the National Cancer Institute, Bethesda, managed to produce bladder cancer in dogs by continuous treatment for several years with large dosages—up to 500 mg. per day—of 2-naphthylamine, one of the suspect dye intermediates. Yoshida in Japan in 1933 observed liver cancer in mice after external application of 4-o-tolylazo-o-toluidine (oaminoazotoluene). Soon thereafter, Kinosita reported that the coloring matter N,N-dimethyl-p-phenylazoaniline (4dimethylaminoazobenzene or butter yellow), once used to dye butter and margarine, gave rise to liver cancer when fed to rats on a vitamin-deficient rice diet. Previously this material had been thought harmless in man and animals because it had no effect in rabbits. About the same time another aromatic amine, 2-fluorenamine (or 2-aminofluorene), was patented as an excellent insecticide by Claborn and Smith of the U.S. Department of Agriculture. Fortunately, prior to commercial use, Wilson, DeEds, and Cox of the USDA Western Regional Laboratory made a study of the chronic toxicity of this material. Perhaps unexpectedly for that time (1940), they found the compound to be a powerful carcinogen, causing a multitude of cancers in various organs of rats and mice. This finding, of course, precluded the use of 2-fluorenamine or the amide, N-2-fluorenylacetamide ( 2-acetylaminofluorene), as an insecticide. On the other hand, all was not lost. The material has been used extensively and perhaps just as valuably in fundamental investigations on the mechanisms of cancer production. Solvents
as Cancer
Problems
N-Nitrosodimethylamine (dimethylnitrosamine), an effective solvent, is also an intermediate in the production of dimethylhydrazine, an important item in the manufacture
of drugs and rocket fuels. However, two employees in the British pilot plant preparing the nitrosamine developed symptoms of early liver disease. Management, concerned by this finding, asked Barnes and Magee of the Medical Research Council of Great Britain to investigate. They reported in 1956 that N-nitrosodimethylamine and related compounds were highly hepatotoxic (toxic for the liver) in animals and also led to tumors of the liver, the kidney, and certain other tissues in rats. Another series of well known commercial solvents includes the halogenated hydrocarbons, especially carbon tetrachloride. In small doses it causes liver damage in virtually all species, including man. In addition, this compound has induced liver tumors in several inbred strains of mice and in hamsters. Considering the toxic effects in all species and carcinogenic effects in at least two species, compounds such as carbon tetrachloride deserve to be handled with utmost care. Dioxane also is a moderately potent fiver carcinogen in rats, as is thioacetamide, used to generate H 2 S in analytical laboratories. The potential hazard from the use of such solvents may be less in industrial areas where close supervision and good safety practices can be exercised than in the household or small shops where inadequate precautions may be taken. The commercial process leading to isopropyl oil gave rise to carcinogenic products, for some employees were afflicted with cancer of the respiratory tract. The processes have been corrected to avoid the dangerous intermediates. Cancer hazards are also associated with some inorganic chemicals, but these are outside the scope of this article. In 1960, the poultry industry in Great Britain was plagued by an outbreak of a mysterious disease, turkey X disease, which killed more than 50% of the poults. The lethal agent responsible came from mold-contaminated peanut meal, which was part of the diet. Subsequent investigations, especially by Allcroft and Carnaghan of the Central Veterinary Laboratory in Weybridge, revealed that the mold Aspergillus flavus produced a toxic factor, aflatoxin. This factor is not only hepatotoxic in animals but it is also carcinogenic upon continuous feeding, according to FEB. 7, 196 6 C&EN
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studies first made by Lancaster of the English Unilever Research Laboratory and confirmed by others. This development gives new insight into the occurrence of liver cancer in man, especially in countries where by habit or necessity mold-contaminated food is consumed. Testing Chemicals for
Carcinogenicity
To be able to make a judicious assessment of structureactivity relationships, discussed later in this article, we must first understand something of the methods used to detect and evaluate carcinogenic effects of chemicals. One important approach is the study of the occurrence of cancer in man exposed to specific products through diet or occupation: for example, liver cancer in tropical countries, or bladder cancer in the dye industry before the hazard had been defined. Another method involves chronic toxicity studies in animals of pharmaceutical or industrial products. Hopefully such studies are made prior to the general application of the materials in commerce, as was the case with N-2-fluorenylacetamide and N-nitrosodimethylamine. A lead to the activity of nitrosodimethylamine was provided by the observation of a noncancerous, yet toxic, response in man. Deliberate tests for carcinogenicity are essentially like those for chronic toxicity. Careful observation of animals of various species, ages, and both sexes, exposed by sev-
CANCER AS AN OCCUPATIONAL DISEASE A t t h e e n d of t h e 1 8 t h c e n t u r y , Sir Percival P o t t , a B r i t i s h physician, linked t h e occurrence of cancer of t h e s c r o t u m in c h i m n e y sweeps t o t h e i r occupation» Small b o y s were chosen for t h i s profession, a n d t h e y were exposed for m a n y y e a r s t o t h e soot a c c u m u l a t e d from t h e b u r n i n g of soft coal- H y g i e n i c practices were poor. However, t h e d e m o n s t r a t i o n t h a t soot or t a r w a s actually responsible for t h i s form of cancer remained unproved u n t i l 1916 when Y a m a g i w a a n d Ichik a w a r e p e a t e d l y applied e x t r a c t s of t a r t o t h e ears of r a b b i t s a n d p r o d u c e d cancer a t t h e p o i n t of application» After World W a r I , B r i t i s h investigators u n d e r t h e leadership of Sir E r n e s t K e n n a w a y , d e m o n s t r a t e d b y persistent a n d tedious exp e r i m e n t a t i o n t h a t cancer could b e induced on t h e skin of mice b y coal t a r . F r a c t i o n a t i o n of t h e t a r t o isolate t h e active principle led t o t h e discovery t h a t t h e t u m o r - p r o d u c i n g cons t i t u e n t s were also highly fluorescent. T h u s , e x a m i n a t i o n of fractions for fluorescence r a t h e r t h a n l e n g t h y a n i m a l t e s t s guided t h e s e p a r a t i o n s , a n d rapid progress w a s made» A brilliant discovery followed shortly: C e r t a i n polynuclear a r o m a t i c h y d r o c a r b o n s , s u c h as t h e n o w familiar b e n z o [a ]pyrene or 3 - m e t h y l c h o l a n t h r e n e , contained in t h e t à r cause, i n p a r t , t h e t a r ' s carcinogenic effect» ,
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eral routes for one to two years or even their entire life (controls are untreated or given vehicle alone, and held under identical conditions at the same time), permits an assessment of relative carcinogenicity of test materials. Microscopic histopathological examinations are needed to confirm the identity of any lesion resulting from the treatment. Observations of this kind permit noncancerous and neoplastic growths to be distinguished. Only this way can tumors be properly diagnosed and classified. The mouse has come to be the classic animal for studies of carcinogenicity. Interest in genetics and the needs of investigators in carcinogenesis have led to the development of strains of inbred mice and rats which are uniform and show a standard response. They are the equivalent in biology of reagent chemicals in chemistry. Dogs have been used because of the first success in simulating the human problem of bladder cancer production by dye intermediates. However, dogs are expensive and difficult to house in large numbers. Also, the time required for a positive response is from two to eight years even with powerful carcinogens. Hence, dogs are helpful only to answer specific questions. More recently, hamsters have aroused interest as test animals. They develop bladder and liver cancers in response to certain azo dyes, aromatic amines, and carbon tetrachloride. Young animals are generally used because they can be exposed for a long period of time. Also, they generally respond better. Newborn animals or infants show even
WHAT IS CANCER? Actually, t h e r e a r e a great v a r i e t y a n d t y p e s of diseases collectively classified u n d e r t h e t e r m cancer. If we a r e t o u n d e r s t a n d cancer, which we can define a s a n a b n o r m a l g r o w t h condition, we h a v e first t o k n o w w h a t n o r m a l g r o w t h is. N o r m a l tissues h a v e specific limitations on t h e i r d e v e l o p m e n t a n d h a v e definite functions. C a n c e r o u s or neoplastic cells, o n t h e o t h e r h a n d , d o n o t h a v e t h e s a m e limitations. R a t h e r , cancerous cells p r e s e n t a p i c t u r e of loss of over-all control b y t h e h o s t organism in respect t o cellular multiplication. Often, such cells show p a r t i a l or complete loss of specialized functions. S o m e t y p e s of cancer cells a c t u a l l y grow v e r y slowly. M a n y n o r m a l tissues r e p r o d u c e themselves a n d divide m u c h m o r e r a p i d l y . H o w e v e r , t h e u n i q u e p r o p e r t y of s t i m u l a t e d cancer tissue in a g r o w t h p h a s e is t h a t it will d i v i d e a n d i n v a d e inexorably, a p r o p e r t y n o t s h a r e d b y n o r m a l tissues.
greater sensitivity, which is explained by the presence in these very young of different metabolic pathways and hormonal status, or of inability to develop an immune response. Some carcinogens are relatively innocuous in a single, even large dose, but may be quite toxic, often increasingly so, upon continued administration. Thus, preliminary toxicity studies must be carried out for at least one month to gather adequate data on tolerated dosages. Long-term observations at a single dose level, if close to or a sizable portion ( 1 / 2 or 2 / 3 ) of the maximum tolerated dose, can yield information on the carcinogenic potential of a compound. However, even more valuable results can be obtained by using several dose levels from which a dose-response curve can be prepared. Smaller doses extend the latent period in addition to lowering the tumor incidence. In some cases, moreover, the specific organ involved may depend on an alteration in dose or regimen of administration of compound. For example, nitrosodimethylamine given continuously leads to liver cancer in rats. If only a few doses are given, on the other hand, the liver is able to meet the challenge. It is only after a longer interval that cancer of the kidney develops. In a similar manner, JV-2fluorenylacetamide induces liver tumors in rats. In the presence of tryptophan or indole derivatives, carcinogenesis in the liver is inhibited somewhat, although bladder cancer results later. In dog and man, the liver may be less sensitive to a
CANCER IS CLASSIFIED BY TYPE • Whereas cancer in humans is of primary interest to most of us, we must recognize t h a t cancer also develops in animals and plants. Neoplasia can be found in virtually every organ, although in some species certain tissues are affected more often, than others. Cancer of the epithelial cells is called a carcinoma, whereas cancer in the connective tissues is classified as sarcoma. .Both kinds of cancer can occur together. In addition, cancer from one tissue can spread to other organs as metastases, which, in man,' are found in many locations such as in lung, liver, bone, brain, and kidney, • A lesion is classified as cancerous or malignant when microscopic examination shows it to be invading neighboring tissues or when metastases are found. Malignant neoplasia are characterized by the fact that transplants of small bits of tissue into similar or even different hosts will grow and reproduce the cancer. Such transplantation in animals was first practiced late in the 19th century. • Other forms of neoplasia are classified as benign because the abnormal growth is localized at the original site. The tumor does not generally grow rapidly although it can do so depending on external conditions. Often it is not transplantable.
carcinogenic challenge such as an aromatic amine than in mouse or rat. Thus, cancer develops in the bladder of dog or man after a longer interval. A chemical that is reliably and definitely carcinogenic in one or two species very likely will be so in other species, including man. The site affected need not be the same, for it depends on metabolic, hormonal, and genetic factors. Recent studies show that man and animals share many biochemical and physiological pathways, and respond alike to exogenous agents. Potent carcinogenic compounds can be identified readily, for when administered by a variety of pathways to virtually any species they will affect some tissue in most animals. With the less potent agents, however, selection of the test species and the route of administration become more important. Also, the number of animals used will have to be larger to get data that have statistical significance. Typically, a compound giving a 30% incidence of tumors in rats or mice would be relatively weak and one giving a 5% incidence would be dubious. On the other hand, such figures would be unthinkably high in the case of man. Of course, man is generally not exposed chronically to high levels of a carcinogen. Carcinogenicity
and Test
Procedures
Some compounds cause cancer at the point of application—for example, polynuclear aromatic hydrocarbons such
CANCER IS FOUND IN VIRTUALLY ALL TYPES OF LIFE Cancer as a disease condition has been known since antiquity. I t strikes m a n and animals a t all ages b u t appears more frequently in older individuals of t h e species. Thus, with t h e ever increasing lifespan of t h e human population it is n o t t o o amazing that cancer continues t o assume an ever more important aspect in t h e diseases affecting man. Cancer does not occur t o an equal extent in all parts of t h e world. Breast cancer occurs with lower frequency in Japan than in t h e U.S. or the countries in Europe. Cancer of the stomach, especially in males, is more common in Japan than in t h e U . S . Cancer of t h e liver is not widespread in the Western Hemisphere b u t accounts for a fair proportion of t h e cancers in t h e B a n t u in Africa and in certain populations in t h e Far East. T h e widely publicized incidence of lung cancer is higher in t h e industrialized world and is increasing a t a n appreciable rate.
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Stimulated cancer tissue in a growth phase exhibits uncontrolled cell multiplication. The liver cells in the left photomicrograph are normal. In the center picture, the liver cells are malignant and rapidly dividing. At right, the lung tissue contains both normal (white) and malignant (dark) cells
as benzo[a]pyrene. Probably, these materials are themselves the actual causative agents. In contrast, the carcinogenic azo dyes, aromatic amines, and nitrosodialkylamines are not generally active at the point of application. Rather, they affect the liver and other internal organs. There is good evidence that these compounds are not themselves carcinogenic, but require biochemical activation—a sort of lethal synthesis. Generally, they must be given in large doses for a long time, because only a small portion is actually metabolized to the proximate or ultimate agent. Consideration of this distinction is important in choosing the proper assay system. It makes no sense to use skin painting for a compound which requires metabolic activation and then to expect tumors in the painted area. Similarly, some of the known carcinogenic hydrocarbons, which are highly effective in microgram amounts when applied to the skin of mice, may be virtually harmless when administered orally because they are rapidly detoxified. With amines, nitrosamines, dyes, and similar chemicals, the oral route, either by admixture in the diet or by stomach tube at regular intervals, is most effective and simulates most closely the potential exposure of animals or people. Application of a test material, either undiluted or with a vehicle, to the skin is a useful test and mimics the exposure of humans in some environments. In general, the skin is amazingly permeable to organic compounds, although water-soluble materials are generally absorbed much less readily. Subcutaneous injection of carcinogenic hydrocarbonseven a single small dose—has led to sarcomas in both mice and rats. Care must be taken in interpreting such results because sarcomas occasionally are found after injection of vehicles alone or of concentrated solutions of water-soluble physiological compounds such as glucose or sodium chloride. The use of several dosage levels of the chemical is advisable. Then, if the response is proportional to the dose, the effect is most likely due to the material tested. A poor dose response renders the data suspect. Although common in earlier days, this method is not much in vogue now with the compounds discussed in this article. Intraperitoneal injection is a simple but effective route for administration. Foreign substances are absorbed fairly readily even if injected as a suspension. Most carcinogens given in this fashion affect the same organs that they do if administration is by an oral route. In addition, if the 128
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compound is locally active, it will produce tumors (sarcomas) within the peritoneal cavity. Inhalation has been tried as a way of exposing experimental animals in studies relating to the lung cancer hazard caused by air pollution and smoking. The equipment required is fairly elaborate, especially for aerosols or dusts. Curiously enough, this procedure often has not been found reliable (except for gases) to demonstrate carcinogenicity, even with materials known to contain active agents. Possibly the mucus in the nasal system of animals can remove dusts and irritating substances. On the other hand, man engaged in heavy work is more likely to inhale through his mouth, thereby exposing the lungs and bronchial system. The smoke from cigarettes likewise reaches the lungs directly. The solvent sometimes plays a role in studies on carcinogenicity. Although the solvent causes little difference in effect when a compound is fed orally, it may cause great differences when certain carcinogens are applied to the skin or are given through intraperitoneal or subcutaneous injection. These differences reside mainly in the relative rates of release of the test material from the solvent to the organism. In general, organic compounds are absorbed more rapidly from an aqueous solution or fine suspension than from an oily vehicle. For skin painting techniques, acetone or toluene are preferred solvents. Although benzene has been used for skin painting, it is metabolized to toxic phenol and thus is not without effect on the host. Toluene by contrast is converted to less harmful benzoic acid. Response and Test
Conditions
With most of the compounds discussed, animals require repeated exposure. In fact, a minimum treatment period is often needed below which any initial effects due to toxicity disappear. However, in contrast to other drugs, carcinogens have been shown to induce permanent alterations in tissue even at subthreshold levels. Subsequent exposures lead to tumors as if interrupted dosing were cumulative. Nevertheless, with a few compounds cancer develops even with a single dose, especially when the compound is tested in newborn animals. (Often, observation in singledose tests continues for a longer time than in the repeateddose tests.) In any case, no conclusions concerning carcinogenicity can be drawn from negative results unless the
Virtually all of the structure-carcinogenic activity relationships discussed in this article were obtained in highly controlled, comparative animal experiments involving mostly mice or rats. Unless specific reference is made to effects observed in man, it should be understood that the
results of animal tests are described. However, extrapolation to man is justified in most cases. Even though the bladder tumors in dye workers were labeled aniline cancers, they were due to the higher homologs. Several independent tests of pure aniline, aniline hydrochloride, or the acetyl derivative, acetanilide, revealed that aniline is not carcinogenic. Continuous ingestion, however, may affect the blood-forming system and lead to blood disorders. On the other hand, recent interest has focused on several di- and trimethylanilines since they are toxic to the liver and preliminary reports indicate some are weak carcinogens in animals. Naphthylamines. The first inkling that 2-naphthylamine might be carcinogenic came from the clinical observations of workmen handling this material. But experimental confirmation came after many negative trials. The early research workers did not realize that continued ingestion of relatively high doses is necessary. In susceptible mice, 2naphthylamine usually leads to liver and bladder cancer. 3-Methyl-2-naphthylamine is a more active carcinogen in animals than the parent structure. Halogen derivatives of 2-naphthylamine have not been assayed for carcinogenicity but experience with similar systems indicates that they should be used with care. Hydroxylated derivatives of 2-naphthylamine are important metabolic products in various animal species and have been thoroughly tested. 2-Amino-6-naphthol is not carcinogenic to animals. On the other hand, 2-naphthylhydroxylamine is active in rats and mice. o-Aminophenols are fairly unstable and readily undergo oxidative decomposition. There is some suspicion that cancer induction by 2-amino-l-naphthol may involve some undefined oxidation product. Highly purified 2-naphthylamine has never given tumors at the point of injection, but rather has affected a remote organ such as the liver or bladder. However, similar injection of an old brownish solution has yielded sarcomas at
Laboratory worker in Dr. Philippe Shubik's lab at Chicago Medical School applies a chemical to the skin of a rat in a test for potential carcinogenicity
Another way to administer test chemicals is by subcutaneous injection. Dr. Yasuhiko Shirasu at National Cancer Institute injects a mouse with a potential carcinogen
test animals are observed for at least one year. Usually tests last about two years for mice, rats, or hamsters, and at least five years for dogs. Under optimum conditions, the most powerful agents rarely give positive indication of carcinogenicity until after three to six months. Well-controlled studies for the lifetime of the test animals are mandatory with weak agents. Reports on safety after subacute, six-month tests are meaningless. Tests in which tumor production is an end point, but in which the latent period is reduced, need refining. For example, species having short life spans, or very young animals, may show enhanced sensitivity. Other ways to increase sensitivity involve the careful selection of enhancing factors such as another chemical, a hormone, or a virus. Kotin and Falk, then of the University of Southern California and now at the National Cancer Institute in Bethesda, have induced lung cancer in mice by using ozonized gasoline vapors and infection with influenza virus, but not with either agent alone. Tissue and organ cultures are also being investigated, especially since embryonic tissues on occasion have shown rapid response. However, these techniques are still in an experimental stage and require thorough evaluation. The aim of any test is to reveal the intrinsic capability of a chemical to induce cancer, ultimately in man. Therefore, strenuous efforts must be exercised to keep this important goal in mind. Aromatic Amines: Structure-Activity
Relationships
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the site. The exact mechanism of this change is not yet understood, although Radomski of the University of Miami has evidence on dimeric intermediates. It indicates, however, that there may be increased hazard with impure preparations such as might be found in technical-grade products, mother liquors, reaction vessels, and even effluent waste.
The isomer 1-naphthylamine, a commercially important material, has been tested for carcinogenicity repeatedly. Initially, some assays seemed to suggest that it was active. However, a thorough study with a pure preparation, free of the 2-isomer, gave negative results. In view of the demonstrated carcinogenicity of 2-naphthylamine this compound is no longer used as a dye intermediate in some countries, including the U.S. It is specifically prohibited in certain states of the U.S. Still unresolved is why 1-naphthylamine and other compounds with this molecular configuration are inactive. It could be either inherent in the structure or a consequence of rapid detoxification through the 4-position. If the reason is detoxification, proper substitution at the 4carbon would block this reaction and give an active compound. Biphenylamines. 4-Biphenylamine (or xenylamine) also was an important dye intermediate and a rubber antioxidant used in the U.S. Some cases of bladder cancer in humans were ascribed to its manufacture and use. At times, however, it has been quite difficult to incriminate exposure to a single chemical for a disease seen 10 to 20 years later. Often the same factory produced several of these compounds, and the employees were rotated in the various departments or plants. Nevertheless, authenticated cases of bladder cancer were recorded in humans exposed to 4-biphenylamine and not to other known carcinogens. Animal tests confirmed the hazard due to 4biphenylamine. Its planned introduced in the chemical industry in Great Britain in the early 1950's was therefore fortunately abandoned.
Walpole and his associates at Imperial Chemical Industries near Manchester, England, have performed extensive studies in rats with substituted 4-biphenylamines or its isomers. Introduction of a fluorine at the 4'-position increases the carcinogenicity significantly, and indeed, gives a compound which affects not only the liver but also the kidneys in males and the breasts in females. Methyl groups in the 3- or 3'-position yield a more powerful agent but reduce or abolish the effect when on the 2position. The 2,3-dimethyl derivative has an unusual action in that it induces tumors of the small intestine. In addition, it affects a number of other sites such as the ear duct, the salivary gland, the bladder, and the mammary gland in females. p-Nitrobiphenyl is also carcinogenic. It can be reduced in vivo to the arylhydroxylamine. This N-hydroxy derivative may be the active intermediate in all these cases, for N-4-biphenylylacetohydroxamic acid, a new metabolite of 4'-phenylacetanilide (4-acetylaminobiphenyl), is a more powerful carcinogen than the parent compound. Thus, 4'-phenylacetanilide, N,N-dimethyl-4-biphenylamine, and 4-biphenylamine exhibited carcinogenicity of a similar magnitude in rats, perhaps because of metabolic conversion to the same proximate agent. The isomeric 3-biphenylamine was somewhat less active in rats than the 4-isomer. 2-Biphenylamine appeared to be practically inactive.
Benzidine or 4,4'-diaminobiphenyl is a useful chemical intermediate, an antioxidant, and also a reagent in clinical chemistry. Unfortunately, it is also a carcinogen in man and animals. With this molecule also, substitution of ad-
ditional methyl groups at certain positions maintains the carcinogenicity in rats or mice. Even extension of the chain by a phenyl group to yield p-terphenyl-4-amine yields a carcinogen.
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Fluorine or chlorine at the ortho position gives the active compounds 2'-fluoiO-4'-phenylacetanilide or 3,3'-dichlorobenzidine. Bromine- or iodine-substituted compounds have not yet been extensively studied in animals because of the higher expense of producing them in large quantity. These larger atoms may yield compounds of lower or no carcinogenic potency, which obviously would have to be checked if they were to be of commercial value.
ferent pattern of sensitivity, of latent period, and even of susceptibility to the toxic effect, an indication of the importance of hormonal factors. Manipulation of the hormonal environment by castration and substitution of the opposite hormone, by hypophysectomy, or by adrenalectomy has demonstrated direct or indirect hormonal involvement in the development of cancer in several organs of rats such as the mammary gland and the liver. Chlorpromazine delays but reserpine accelerates liver carcinogenesis by N-2-fluorenylacetamide by a similar modus operandi. We have shown that pituitary hormones powerfully reinforce the carcinogenicity of N-2-fluorenylacetohydroxamic acid, the N-hydroxy metabolite of N-2-fluorenylacetamide.
Separation of the two rings in biphenylamine by a —CH=CH— grouping leads to 4-stilbenamine, which is highly active in low doses. The compound is notable since it produces many ear duct tumors in rats.
N-2-Fliiorenijlacetamide (2-acetylaminofluorene) and related compounds. Of all the carcinogenic aromatic amines, N-2-fluorenylacetamide is by far the most studied in animals. Structure-activity studies of related agents; the effects of species, strain, sex, age, dietary and hormonal environment, and metabolism; and the influence of N-2-fluorenylacetamide on a variety of host factors and body constituents have been investigated. We can but briefly summarize some of the salient facts, which serve as a model for this type of agent. N-2-Fluorenylacetamide is a more powerful carcinogen than the corresponding naphthyl and biphenyl derivatives. Moreover, this compound evokes neoplasia in many different organs, in part depending on species and strain. However, it has never done so at the point of application, leading to the conclusion that the compound itself is not the active agent but is metabolized to one. In male rats the liver is often the primary target; in females, the mammary gland. Ear duct and intestinal tumors are fairly frequent. In some strains or species in which liver or mammary glands are less sensitive, organs such as the bladder are also affected, but more slowly. Thus far only two species, the guinea pig and the steppe lemming, of the many studied are resistant to this carcinogen because they do not form an active metabolite. Usually diet has little effect on the carcinogenicity of N2-fluorenylacetamide. Varying the vitamin, protein, and fat levels has sometimes altered slightly the latent period or the susceptible organ but has not changed significantly the over-all response. Male and female animals show a dif-
Administration of N-2-fluorenylacetamide with other compounds has led to interesting developments. Thus, dietary tryptophan, indole, and related compounds appear to give partial protection to the liver, as evidenced by a longer latent period. In a sensitive strain such as the Fischer or Wistar rat, bladder tumors are then seen. Treatment with a mixture of N-2-fluorenylacetamide and a hepatotoxic agent such as carbon tetrachloride enhances responsiveness of the liver, so that more tumors are produced in a shorter time. Mixtures of N-2-fluorenylacetamide and nitrosodialkylamines have the same effect. However, some years ago research workers discovered without apparent explanation that the classic skin carcinogen, methylcholanthrene, fed together with N-2-fluorenylacetamide inhibited carcinogenesis. A rational explanation is now available—methylcholanthrene stimulates the biochemical conversion of N-2-fluorenylacetamide to inactive detoxification products. As in the case of naphthylamine and biphenylamine, the 1- and 4-fluorenylacetamides are virtually inactive; N-3fluorenylacetamide is weakly active. Alkylation of the amino group to give 2-methyl- or dimethylfluorenamine alters somewhat, but does not abolish, the carcinogenic response in rats. The higher acylamides have not been examined for carcinogenicity. They should be active, since Gutmann at the Veterans Administration Hospital in Minneapolis has described enzyme systems which can deacylate them. On the other hand, the slowly hydrolyzed amides from benzoic acid or phthalic acid are weak carcinogens, while the p-toluenesulfonamide which does not hydrolyze is inactive. Thus, it can be concluded that an active carcinogen results only if the amino group can be freed metabolically. FEB.
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compound. Oxidation of the methylene group at the 9position to a keto or hydroxy group, or replacement by oxygen or sulfur, also decreases or modifies the carcinogenicity to some extent in animals. Interestingly, the compound with oxygen at the 9-position, namely 3-aminodibenzofuran (2-aminodiphenyleneoxide), and more so its 2-methoxy derivative, gives mostly bladder tumors. A methoxy in the 7- or the 1-position reinforces the carcinogenicity of N-2-fluorenylacetamide. Ring methyl derivatives of 2-fluorenamine have not been studied, but by analogy with the naphthyl- and biphenylamine they should also show enhanced carcinogenicity.
When added to the diet, 2-nitrofluorene which is a little less carcinogenic than N-2-fluorenylacetamide gives lesions of the forestomach in rats in addition to the customarily found tumors. 2,7-Dinitrofluorene, even though it is relatively insoluble, was a more active carcinogen than the more soluble 2,5-dinitro isomer. In addition, A^N'-fluoren2,7-ylbisacetamide (2,7-diacetylaminofluorene) is a more powerful carcinogen than N-2-fluorenylacetamide, inducing a great variety of tumors in rats, including a few of the difficult-to-induce cancers of the glandular stomach. N,N'Fluoren-2,5-ylbisacetamide is less potent than N-2-fluorenylacetamide and acts chiefly on the mammary gland and ear ducts.
Substitution of alkyl groups at the 9-position of fluorene increases the molecular thickness and results in an inactive 132
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Substitution by fluorine at most positions on the ring maintains the carcinogenic activity in animals and has a potentiating effect at the 7-position. James and Elizabeth Miller of the University of Wisconsin have employed substitution by fluorine as a clever tool for determining the positions which react with the tissue constituent involved in the carcinogenic process. The fact that all of the fluorinated derivatives of N-2-fluorenylacetamide studied thus far are carcinogenic to rats suggests that the ring positions 1, 3, 5, and 7 do not participate in the interaction of the carcinogen with a tissue receptor. Chlorine substituted at the 7-carbon also seems to enhance the carcinogenicity. On the other hand, iodine with a larger atomic radius, when at the 7- but not at the 3-position, appears to eliminate carcinogenic properties.
Assays of the various ring hydroxylated compounds such as 1-, 3-, 5-, and 7-hydroxy-2-fluorenylacetamide, which are metabolites in most species, have shown that they were much less active, or in fact inactive, in animals. Higher aromatic amines. 2-Anthramine is both a potent and highly unusual carcinogen. In contrast to other aro-
matic amines, it not only produces tumors at the expected sites—liver, bladder, and the like—but upon application to the skin it also yields carcinomas. Thus, it acts dually, as a remote liver carcinogen and also at the point of applica tion in the fashion of the classic carcinogenic polynuclear aromatic hydrocarbons. This behavior at first sight ap pears unusual. The explanation may reside in the molecu lar features of this compound. Indeed, it may cause liver tumors in animals because of metabolic conversion of the amino group to the active hydroxylamine. The skin tumors at the point of application may be due to activation of the 3-position or the 3,4-bond (K-region?) by electron transfer from the amino nitrogen so that 2-anthramine reacts like one of the carcinogenic hydrocarbons. Similar data have been reported recently for other polynuclear aromatic amines.
Both 2- and 3-phenanthreneacetamides as well as the 9,10-dihydro derivative are active carcinogens in rats. The 1- or 4-substituted amino derivatives of either anthracene or phenanthrene have not yet been examined, but it is prob able that they would be inactive. N-1-Pyrenylacetamide has only low activity. Metabolism of Carcinogenic Aromatic
Amines
Because most of the carcinogenic aromatic amines were not active at the point of application, research workers sus pected that these compounds required metabolic activation. Thus, considerable efforts were exerted to discover the ac tive metabolite, the "real carcinogen." Years of study and a great variety of techniques were brought to bear on this problem. Metabolic dealkylation or deacylation is essential to re• veal carcinogenicity of ^-substituted amines. Virtually all animal species, as well as man, have enzyme systems in ' many tissues which can remove the acetyl groups from Nacetylarylamines or oxidatively liberate the arylamines from the N-dimethyl or N-methyl compounds. Thus, in such cases where acetyl derivatives or N-methyl derivatives are tested in animals little difference can be seen in their carcinogenic potency. Variations which were encountered were probably due to relative rates of resorption or pref erential destruction. For studies on carcinogenicity, the more stable acetylamino derivatives are usually preferred, especially when the compound is to be mixed in the diet. In this case, storage and exposure to a large surface might lead to decomposition products when the amines are used. All species except dogs can acetylate aromatic amines via the reactive acetyl coenzyme A so that an equilibrium between amines and their acetyl derivatives exists in vivo. N-Methylation of arylamines, on the other hand, is not a significant pathway.
Biochemical ring hydroxylation often leads to inactive, rapidly excreted aminophenols. With most aromatic amines, biochemical hydroxylation on the aromatic ring was the usual finding. For example, mice, rats, and dogs excrete 2-amino-6-naphthol and 2-amino-l-naphthol after a dose of 2-naphthylamine. Likewise, 4 , -phenylacetanilide appears to yield mostly the 4'-hydroxy derivative. N-2Fluorenylacetamide is converted into various ring-hydroxylated derivatives, chiefly at the 5- and 7-carbon atoms. These compounds are excreted in small amounts as free compounds, but the major part is conjugated with glu curonic acid and, in some cases, with sulfuric acid. Dif ferent species form varying proportions of the ring-hydroxylated compounds which suggests that perhaps more than one enzyme system performs these reactions. Tests of these ring-hydroxylated metabolites for carcinogenicity indicate that they are less active than the aromatic amine itself. They are mostly detoxification products, produced by a defense mechanism to eliminate harmful materials, ex plaining why introduction of certain substituents (CH 3 —, CI—, F—, —OCH 3 ) at ring positions needed for detoxifi cation usually gives more active carcinogens. Biochemical Ν-hydroxylation is apparently essential to convert amines to the active carcinogenic intermediates. In 1960, James and Elizabeth Miller and their associates at the University of Wisconsin noted a new hydroxylated metabolite of N-2-fluoreny lacet amide. Patient and skillful isolation of this unknown compound eventually revealed it as the N-hydroxy derivative. When this derivative was tested for carcinogenicity, it was more active than the parent structure and it also caused cancer at the point of application. This activation reaction, N-hydroxylation, then appeared to lead to the long sought-for carcinogenic intermediate. After this initial success, it was quickly found that vari ous other carcinogenic aromatic amines also undergo Nhydroxylation. Furthermore, the study of various species and strains of animals indicated that N-hydroxylation was performed with unequal facility. The guinea pig and steppe lemming either do not have the specific enzyme to
FEB. 7, 196 6 C&EN
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carry out this step or else have large amounts of an enzyme performing the reverse reaction. Man, however, can Nhydroxylate N-2-fluorenylacetamide. N-Hydroxylation ap peared to increase during the feeding of an amine, so that the portion of a dose converted to the N-hydroxy deriva tive became larger. Growing or regenerating tissue seemed to have more of the N-hydroxylating enzyme than resting tissue. Thus, the damage produced by a small amount of this compound led to production of more of it. Administration of methylcholanthrene and certain other polynuclear hydrocarbons enhanced enzymes specific for ring hydroxylation (detoxification) and did not affect, to a great extent, the system required for N-hydroxylation. N-2-Fluorenylacetohydroxamic acid ( N-hydroxy-2acetylaminofluorene) or similar N-hydroxylated N-acetylarylamines are probably not the actual, proximate agents responsible for carcinogenicity. The term proximate agents refers to those chemical structures which interact with specific cellular and molecular targets to produce the neoplastic cell. Studies are under way in many labora tories to unravel further this complicated but crucial prob lem. In any case, the data so far produced convincingly demonstrate that carcinogenesis by aromatic amines some how involves the N-hydroxylated intermediate.
Carcinogenic Azo Dyes Because dyes are used in food, clothing, and cosmetics and, therefore, touch us in our daily lives, the dyes that we use for such purposes must be harmless. Fortunately, most of the commercial dye molecules have polar substituents that make them innocuous. Number of carcinogenic azo dyes limited. Only a few azo dyes are carcinogenic in animals, mostly to the liver. One of the first active compounds discovered was 4-o-tolylazo-o-toluidine. The position of the methyl group in the basic structure affected the carcinogenicity con siderably. Most effective was the p-phenylazoaniline molecule with a methyl group at the 2- and 2'-positions. Moving the amino or methyl groups to other positions re duced the activity somewhat but still gave carcinogens.
just as N-2-fluorenylacetamide has received major atten tion in the aromatic amines, Ν,Ν-dimethyl-p-phenylazoaniline (butter yellow) is the azo dye used most for animal studies. This carcinogen usually affects the liver in rats, mice, hamsters, or dogs but is inactive in guinea pigs. At least one methyl group on the nitrogen is required for ap preciable activity, but no one has discovered why. Ring-substituted derivatives of N,N-dimethyl-p-phenylazoaniline can be either more or less active than the parent structure depending on the substituent and its location. Hydroxy groups invariably abolish carcinogenicity. On the other hand, addition of methoxy enhances carcinogenicity, alters the target, and abolishes the need for N-methyl substitution. Ν,Ν-Dimethyl-p-phenylazo-o-anisidine (3methoxy-4-dimethylaminoazobenzene) causes mammary, ear duct, and even skin cancers in rats instead of affecting the liver only. Substitution at the 3'- or 4'-position by alkyl or by halogen generally increases the activity. The effect on the 2'-position varies with the substituent. Steric factors, such as the halogen being ortho to the azo link, may play a role. Addition of substituents on the ring bearing the dimethylamino group generally results in a lowered ac tivity, except with fluoro or methoxy groups. Polar sub stituents such as sulfonic acid groups commonly abolish the carcinogenicity, probably because of an increased rate of excretion or impermeability of the cell membranes. Active compounds result when mono- or polysubstitution of fluorine takes place on all ring positions. The ex ception is the 3,5-difluoro derivative, in which positions ortho to the azo bond are blocked. This exception suggests that the ortho position is involved in the combination of the active carcinogen with the cellular target.
Patent and Technical Literature Makes Many References to the Uses of Carcinogenic Aromatic Amines
Amine
Use Optical bleach ing agent
Belgium
1962
Chloro-2-naphthylamine
Flame-resistant polyolefins
Germany
1962
AT,iV-Dimethyl-4aminobiphenyl
Redox indicator
*
1963
Benzidine
Polymer
U.S.S.R. China England
1963 1963 1962
Ν,Ν,Ν',Ν'-Tetraalkylbenzidines
Polyesters
Japan
1963
*
1963
3,3 '-Dichlorobenzidine Elastomers
7, 196 6
1963
Germany
1963
Polymers
Germany
1963
iV,A/r,-Dialkyl-3,3/dimethoxybenzidine
Heat- and lightresistant fibers
Japan
1963
*
1963
* Literature
FEB.
*
3,3 '-Dimethoxybenzidine
Analytical re Ν,Ν,Ν',Ν'-Tetraagent methyl-3,3 '-dimethoxybenzidine
C&EN
Date
2-Naphthylamine
3,3 '-Diaminobenzidine Analytical re agent Polymer
134
Patents or Proposal
For reasons that are not clear, the carcinogenicity of N,N-dimethyl-p-phenylazoaniline is lost when both N-methyl groups are replaced by higher alkyls such as ethyl or propyl. However, insertion of an ethyl in the 4'-position restores the activity of IV-diethyl derivatives. Although replacement of one of the phenyl rings of N9Ndimethyl-p-phenylazoaniline by another ring system such as 1- or 2-naphthyl yields active compounds, a pyridine or quinoline ring leads to considerable enhancement of the carcinogenicity. For example, 5- or 6-(p-dimethylaminophenylazo) quinoline-1-oxide are among the more po tent compounds of this class in rats.
Carcinogenicity of azo dyes does not necessarily require an amino group on the ring since l-o-tolylazo-2-naphthol and 2,2 r -azonaphthalene, but not the 1,1'- or the l,2'-isomers, are active in rats and mice.
Regulation and safe practices for dyes. As a result of the responsibilities of the Food and Drug Administration or its counterparts in various countries, dyes have been examined with respect to their safety, particularly from the viewpoint of carcinogenicity. In the course of such in vestigations some dyes were found carcinogenic in animals by several adequate and reliable tests. There are, though, no proved cases of cancer in man due to occupational or dietary exposure to the azo dyes. Several of the dyes are fairly large molecules containing substituents which in smaller molecules, such as N,N-dimethyl-p-phenylazoaniline, would lead to inactive products. Thus, substitution by a highly polar sulfonic acid residue virtually always leads to inactive compounds with the smaller azo dyes, aro matic amines, and even the carcinogenic hydrocarbons. However, liver tumors have been induced in rats by com pounds such as Ponceau 3R—the azo dye derived from 2',4',5'-trimethylaniline and R acid, 2-naphthol-3,6-disulfonic acid—and by Trypan blue.
Trypan blue has been examined for carcinogenicity sev eral times in animals with variable results. The commercial product consists of a mixture of isomers. However, we do know that purified Trypan blue is carcinogenic. It does not give the tumors of the bile ducts or liver cells usually found with azo dyes. Rather it causes sarcomas of the connective tissue of the liver. Combined treatment with Trypan blue and Ν,Ν-dimethyl-p-phenylazoaniline induces both types of liver tumors, indicating that these compounds act independently. Few studies have been performed to understand the mechanism of action of the more complex azo dyes. Where the azo dyes are derived from poly alkylated anilines, 2-naphthylamine, or S^'-dimethylbenzidine (from Trypan blue) investigators have postulated that enzymic reduc tion leads to carcinogenic split products. Dietary and hormonal factors play substantial roles in azo dye carcinogenesis. In contrast to what was found with the carcinogenic aromatic amines, diet exerts a crucial effect on the carcinogenicity of N,Af-dimethyl-p-phenylazoaniline. Indeed, shortly after the discovery of the carcino genicity of this dye by Kinosita in Japan, the same experi ment was a complete failure in the hands of European workers. Kinosita's rats were fed a poor diet of polished white rice with virtually no additional nutrients. The rats in the European experiments, on the other hand, were on a nutritionally adequate regimen. The discrepancy is now understood. There are detoxi fying enzymes in mammalian systems which oxidize the N-methyl, hydroxylate the aromatic rings, and reductively split the azo bond. The last reaction system requires flavine-adenine-dinucleotide as cofactor and operates at minimum levels in animals on a riboflavine deficient diet. With sufficient vitamin this enzyme rapidly converts N,Ndimethyl-p-phenylazoaniline to the corresponding inactive split products, Ν,Ν-dimethylaniline and p-phenylenediamine. Some substituents in the azo dye molecule affect F E B . 7, 1 9 6 6 C & E Ν 135
the rate of reductive splitting. Therefore, groups such as a 3 r -methyl lead to carcinogens which are more resistant to deactivation. Other agents inhibiting the carcinogenic response to azo dyes, but not to N-2-fluorenylacetamide, are salts of cobalt, nickel, or especially copper, and compounds such as p-hydroxyaceto- or p-hydroxypropiophenone and certain benzimidazoles. As with aromatic amines, the carcinogencity of the azo dyes is dependent on the hormonal environment. Thus, removal of the adrenals or pituitary nullifies the carcinogenic effect. Administration of either cortisone or reserpine increases carcinogenicity whereas chlorpromazine moderates it. The operating mechanism may be based on an effect on the enzymes detoxifying the dye, and on the alteration of the host response to the carcinogenic challenge. Metabolism of azo dyes yields several products. The search for the active agent with the carcinogenic azo dyes has thus far yielded ambiguous leads. 4-o-Tolyl-otoluidine and related compounds, which are active without alkylation of the amino group, may be carcinogenic because of the presence of the amino group, with the remainder of the molecule providing the necessary steric features. However, definite information is lacking. In any case, a few metabolic studies performed with 4-o-tolyl-o-toluidine indicate that the azo link is reduced as in other azo dyes. Metabolic hydroxylation of the rings and of the nitrogen (significant for carcinogenic potency) or oxidation of the methyl groups to carboxy probably occurs.
Carcinogenic
Nitrosamines
From a fundamental point of view one of the most interesting class of chemicals is the nitrosamines. Many of the nitrosamines are both water and lipid soluble. On a molar basis they are more potent carcinogens than most aromatic amines or azo dyes. Despite the fact that organisms can excrete the major part of a dose very rapidly, several of these compounds have caused cancer in rats after a single exposure. The effectiveness of the high carcinogenicity of nitrosamines could not have been predicted on the basis of their structure, considering the state of the art in 1954. However, certain structures, even though possessing a nitrosamine function, are not active. These include diphenyl-, di-tert-butyl, and methyl-ieri-butylnitrosamine. iV-Nitroso-N-methylaniline, however, is carcinogenic. In rats, nitrosodimethyl- and diethylamine and the symmetrical higher homologs generally affect the liver. Kidney tumors result with some of the chemicals, depending on dosage. Also, the diethyl compound secondarily induces cancer of the bladder and esophagus, and the diamyl derivative injected intravenously gives rise to lung, but not to liver, tumors. Unsymmetrical nitrosamines such as vinylethyl, benzylmethyl, and the nitroso derivatives of morpholine, piperidine, and other heterocyclic bases are remarkable in that they mostly affect the esophagus of rats, causing a lesion not often found experimentally in tests with most other carcinogens. On the basis of theoretical considerations of the mode of action of the nitrosodialkylamines, research workers recently tested diazomethane. Tested by inhalation the material gave rise to lung tumors in mice and rats. Chemists, therefore, should avoid exposure to diazomethane not only because of its well-known toxic nature, but more importantly because of its carcinogenicity. Methylnitrosourea,
Many Patented Nitrosamines Are Hazardous Patent In the case of N,N-dimethyl-p-phenylazoaniline, ringhydroxylated products, amines from the reduction of the azo bond, and dealkylated metabolites have been found. However, no clue to their carcinogenicity exists. In view of the importance of N-hydroxylation with the aromatic amines, interest has recently been focused on oxidation of the nitrogen in N,N-dimethyl-p-phenylazoaniline, yielding the corresponding N-oxide. Indeed, Terayama in Japan reports that this compound is a metabolite as active in rats as the azo dye itself. However, conclusive evidence leading to a clarification of the mode of action of the carcinogenic azo dyes remains a challenge.
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(Country of Origin
Date
Intermediates Solvent, intermediate
U.S.A. England U.S.A. England U.S.A. England U.S.A. England England U.S.A. England U.S.A. England U.S.A. France
1963 1958 1963 1958 1963 1958 1963 1961 1958 1963 1958 1963 1958 1963 1962
iV-Nitroso cyclic amines Bactericide Insecticide
U.S.A. Germany
1963 1960
N- Alkyl - Af-arylnit rosaminesi Insecticide Rubber additives Insecticide
U.S.A. Japan Germany
1963 1961 1960
Dialkylnitrosamines Gasoline and lubricant additives Antioxidants Stabilizers Rubber additives Insecticides Fungicides Bactericides
methylnitrosourethane, and related compounds that classically had been used to prepare diazomethane are similarly hazardous. In rats they act on the upper intestinal tract, liver, kidneys, and brain. They have even produced carcinoma of the glandular stomach in rats, histologically similar to the spontaneous stomach cancer of man. Noncarcinogenic N-methyl-N-nitroso-p-toluenesulfonamide can adequately substitute as a source of diazomethane where this is required. In addition to causing cancer in mice, rats, and hamsters, nitrosamines also readily evoke liver tumors in somewhat less sensitive larger species such as rabbits, dogs, guinea pigs, and even monkeys. These findings were remarkable because hepatomas had never before been induced in the latter two species with azo dyes or aromatic amines. Metabolism of nitrosamines gives reactive intermediates. The parent compound, nitrosodimethylamine, undergoes a number of rapid biochemical conversions. The methyl groups are oxidized in part to COL, via the classical onecarbon intermediates, and some of the nitrogen enters the proteins and nucleic acids. A possible metabolic product, dimethylhydrazine, is noncarcinogenic, ruling it out as an active intermediate.
Some Nitrosamines Are Known to be Carcinogenic in Animals R-N-R' 1 1 NO R R' Normal dialkylnitrosamines JV-Ethyl-iV-nitroso-ra-butylamine iV-Methyl-iV-nitrosovinylamine iV-Ethyl-iV-nitrosovinylamine iV-Methyl-iV-nitrosoallylamine iV-Methyl-iV-nitrosoaniline iV-Methyl-iV-nitrosobenzylamine iV-Methyl-iV-nitrosoacetamide 1 -Methyl-1 -nitrosourea Methylnitrosoethyl carbamate iV-Nitrosodiethanolamine iV-Nitrososarcosine ester
ra-H-CCH^
n-H-(CH,)M
C2H5
71-04X19
CH 3
CH2=CH
C2H5
CH2=CH
CH 3
CH 2 ==CH-CtÏ2
CH 3
C&Hb
CH 3
C6H5CH2
CH 3 CH 3
CH3CO NH 2 CO
CH 3 CH 2 OHCH 2 CH 3
C 2 H 5 OCO CH 2 OHCH 2 C 2 H 5 OCOCH 2
Cyclic Nitrosammes 1 -Nitrosopiperidine 1,4-Dinitrosopiperazine 4-Nitrosomorpholine According to present concepts, the activity of the nitrosodialkylamines hinges on the oxidation of one of the alkyl groups. The oxidation leaves a highly reactive, unstable monoalkylnitrosamine, which decomposes to a diazoalkane, which is a typical alkylating agent. Alternatively, the occurrence of a carbonium ion intermediate has been postulated by some workers on the basis that cysteine and similar trapping agents have lowered toxic effects. In any event, the carbon from nitrosodimethylamine is incorporated into liver proteins and into the 7-position of guanine in RNA. Cancer induction might result from the alteration of these fundamental molecules which are concerned with the information .transfer from DNA. Additional experiments regarding the mode of action of this type of carcinogen is currently engaging the attention of many laboratory workers.
Nitrosoanabasine
Dinitrosodiamines iV,iV'-Diethyldinitrosoethylene diamine iV^'-Dimethyldinitrosopropylenediamine Noncarcinogenic or Weak Nitrosamines
Mycotoxins: Powerful, Mold- or Plant-Produced Carcinogens
Diphenylnitrosamine Ethyl-teri-butylnitrosamin( Dibenzylnitrosamine Diisopropylnitrosamine (weak) iV-Nitroso-iV-methyl-ptoluenesulfonamide
Aflatoxin. Subsequent to the outbreak of a strange disease in turkey poults, ducklings, pigs, and calves in England, research workers exerted strenuous efforts to discover the underlying causes. Day-old ducklings served initially as an assay tool for fractions of the suspected diets. An alcoholic extract of diet gave several biologically active spots on thin-layer chromatograms with a blue (B x and B 2 ) or green (Gj or G 2 ) fluorescence. Extensive studies in a number of species showed that the products isolated were not only toxic but also potent carcinogens.
The agents responsible came from contamination of some of the diet ingredients, such as peanut or cottonseed meal, with the mold Aspergillus flavus and followed improper harvest or storage procedures. Structural analysis by van der Merve and co-workers at C.S.I.R. in South Africa, Buchi's group at MIT, and van Dorp's team at the Dutch Unilever Laboratories showed that the mold products responsible FEB. 7, 1 9 6 6 C & E N
137
were 5-membered polynuclear heterocyclic compounds containing only oxygen, hydrogen, and carbon. An amazingly short time elapsed between the discovery of the original disease and the isolation and identification of the complex chemical agent responsible. It is one of the major achievements of natural product chemistry in recent times. There are two main aflatoxins, labeled Bx and Glf of which the former is more toxic and carcinogenic. The dihydroaflatoxins (B 2 and G 2 ) are less active. The specific structural elements responsible for carcinogenicity are not yet known. The activated α,β-unsaturated lactone is one aflatoxin which may serve, perhaps, as an alkylating group. Some lactones, including β-propiolactone, and certain
reaction. Thus, mammalian systems do not have the en zymes necessary to split the aglycone
from the glycoside. The bacterial flora in a normal animal or in man can liberate the active methylazoxymethanol.
From the brief description of these natural products we can see that man does not have the sole responsibility for spreading carcinogens in this world. Molds and plants provide considerable assistance and, indeed, may be re sponsible for a large number of cases of human cancer in areas (Africa, Guam, and most of the countries in the Far East) where unknowingly and by local custom diets containing such potent toxins are consumed. Mechanism of Action of Chemical
epoxides are carcinogenic. The aflatoxins show an un usually high propensity for the liver when fed to test animals as part of their diet. But when injected subcutaneously the aflatoxins cause local sarcomas. Indeed, the aflatoxins are very efficient carcinogens. Research workers have calculated that liver cancer in rats is in duced by a daily dose of about 9000 micrograms of Ν,Ν-dimethyl-p-phenylazoaniline, 750 micrograms of nitrosodimethylamine, but of only 10 micrograms of aflatoxin Bv An extensive outbreak of liver cancer was found several years ago in rainbow trout both in the western U.S. and in other areas of the world. In these instances, the causa tive factors pointed also to dietary agents, either a lipid constituent or some contaminant in the cottonseed meal portion of the pelleted diet (aflatoxin). Yellow rice, cycasin. Among other mycotoxins is the one that is related to yellow rice in Japan. Although the active agent has not been pinpointed, a colorless chlorinecontaining peptide and a yellow pigment, luteoskyrin, seem to be involved. In the Far East and especially in Guam, the local in habitants consume foodstuffs derived from the cycad nut, Cycas circinalis. If the nuts are not properly processed, a highly toxic principle, cycasin, can be present. A cycasincontaining meal manifests itself by causing short-term hepatotoxicity and eventually liver cancer in rats. The agent responsible has been shown to be a glucoside of methylazoxymethanol, a close relative of nitrosodimethylamine with a comparable active intermediate. Laqueur at NIH noted recently that when cycasin is fed to germ-free rats they do not evidence the hepatotoxic or carcinogenic 138
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Carcinogens
Many proposals have been made in attempts to de cipher the causes of cancer, each being a reflection of the proposer's experience. C. Heidelberger of the University of Wisconsin has aptly stated that "our theories are the mirrors in which we see ourselves." The carcinogenic process can be described in terms of a derailment at one or several stages of normal growth and differentiation. Substantial advances in our comprehension of these fundamental phenomena have occurred in recent years. For this reason, it has become somewhat easier to visualize the potential areas where a carcinogen could pos sibly exert its effect. Although there is no universal agreement about the feasi bility of separating the formation of a cancer into separate
Dr. John H. Weisburger is chief of the Carcinogen Screening Section of the Carcinogenesis Studies Branch (Na tional Cancer Institute, National In stitutes of Health). He had been a postdoctoral fellow (1949-50) and a commissioned officer in the U.S. Public Health Service (1950-61) at NCI. Born in 1921 in Stuttgart, Germany, Dr. Weisburger studied at the Univer sity of Brussels and the University of Havana. He received his B.A. in chemistry, however, from the University of Cin cinnati (1947). He also received his M.S. (1948) and Ph.D. (1949) there in organic chemistry. Dr. Weisburger is a member of the American Association for Cancer Research, ACS, and the American Society of Biological Chemists. He also served as an associate editor of the Journal of the Na tional Cancer Institute and was on the editorial board of Cancer Research.
Ordinarily innocuous chemicals can promote tumor growth
INITIATION
PROMOTION
No initiator (7, 12-dimethylben2:[a3-afithracene)
No tumors (control)
initiator, but ho promoter
No tumors (control)
Initiator and repeated doses of promoter
steps, there is experimental evidence that for some types of cancer there are discrete and distinct events which ultimately result in cancer. These have been identified by a variety of names: • Initiation. The initial reaction of the active carcinogen metabolite and its specific target in a cell or tissue, thus giving rise to an abnormal yet microscopically undistinguishable latent tumor cell. • Development. The multiplication of these abnormal cells resulting in visible tumor tissue. It seems reasonable to assume that this step would be subject to different environmental conditions from those required for the initiation step. • Progression. Additional fundamental alterations in the nature of some types of tumor cells.
Dr. Elizabeth K. Weisburger works with her husband at the Carcinogen Screening Section of the National Cancer Institute (NIH). Married in 1947, she has worked with her husband continuously since 1949, first as a postdoctoral fellow at NCI, then as an officer in USPHS, and finally at her present location. They have jointly published nearly 40 papers. Mrs. Weisburger graduated cum laude with a B.S. in chemistry from Lebanon Valley College (Annville, Pa.). She received her Ph.D. in organic chemistry from the University of Cincinnati in 1947. Dr. Weisburger is a fellow of the American Association for the Advancement of Science, the Ohio Academy of Science, and belongs to ACS, Sigma Xi, Sigma Delta Epsilon, Iota Sigma Pi, and the American Association for Cancer Research. She has been working as an abstractor for Chemical Abstracts since 1949.
Jumtm
Initiation:
The Birth of Cancer Cells
The initiation reaction is crucial because it theoretically involves the change of a normal cell to a tumor cell. Experimental evidence for such a step was noticed first some 20 years ago by Berenblum, now at Weizmann Institute, Israel, and Shubik, now at Chicago Medical School, but then at Oxford University in Great Britain. A single minute dose of a carcinogenic polynuclear aromatic hydrocarbon such as benzo[a]pyrene or 7,12-dimethylbenz[a]anthracene applied to the skin of mice shows no untoward reaction unless further treatment is made. The area painted appears normal by all morphological criteria. However, when this area is treated repeatedly with a noncarcinogenic agent, croton oil—even up to one year later— papillomas and carcinomas appear. Thus, skin that is treated once with a small dose of a hydrocarbon undergoes a permanent invisible change. This is termed initiation; the subsequent painting with croton oil to reveal and develop tumors is called promotion or development. Subsequent biochemical studies in this system did indicate that a small portion of the hydrocarbon is firmly bound to certain elements in skin, although the exact nature of this interaction is not yet known. Carcinogenicity
and Binding to Proteins
Extension of the concept of protein-binding to other conditions, both with respect to the chemical carcinogen and the target tissue, has not been easy. The compounds discussed in this report, namely the aromatic amines, the carcinogenic azo dyes, and the nitrosamines, generally do not produce cancer after a single small dose. Moreover, any cancer that is produced, being internal and therefore less easily visible than skin cancer, cannot be studied as readily. In 1947 James and Elizabeth Miller at the University of Wisconsin first noted that in rats treated with N,N-dimethyl-p-phenylazoaniline ( 4-dimethylaminoazobenzene, butter yellow) some of the dye seemed firmly bound to the FEB.
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livers and could not be removed by various extraction pro cedures. Only hydrolysis of the liver proteins released the bound dye. They observed bound dye only in the target tissue, namely the liver in rats, and not in the liver of re sistant species. Dyes of different carcinogenic potency bind at a rate somewhat proportional to their effect, they noted. The tumor induced by a dye, however, did not give evidence of any bound carcinogen. Therefore, they theorized that pro teins capable of combining with dye were absent in the tumor. Since cancer is a form of uncontrolled growth, they reasoned further that the proteins absent from the tumor and present in normal liver had some growth-control functions. The concept of deletion of growth-controlling proteins during the carcinogenic process is based on this experi mental evidence. Sorof, at the Institute for Cancer Re search, Philadelphia, has actually isolated some of the azo proteins by free and column electrophoresis and has under taken to characterize them, a difficult task. This approach was extended to other carcinogens, such as the carcinogenic aromatic hydrocarbons, amines, alkylat ing agents, virtually all of which bind firmly to proteins. However, in contrast to the azo dyes, this binding also oc curs in tissues other than the susceptible organs. Such binding occurs when carcinogenicity in a potential target organ is -inhibited by manipulation of the hormonal environ ment. It also occurs with some noncarcinogenic analogs. However, all carcinogens studied except one (the tricycloquinazoline recently discovered by Baldwin, at the Univer sity of Nottingham, England) combine with protein. The actual carcinogenic metabolite bound or the protein in volved is not yet known for any of the agents, despite considerable efforts. Gene Action and Cancer Recent years have witnessed revolutionary developments in our understanding of the fundamental events in bio chemistry and molecular biology. Whereas the crucial function of nucleic acids in cells was first hesitantly pro posed some 20 years ago, we now have reached the stage where the chemical structure of a nucleic acid is completely known. Imaginative theoretical developments, first applied to microbiological systems but later extended with some modifications to mammalian systems, have in creased our understanding of the operation of various building blocks in a cell. Research workers visualize deoxyribonucleic acid (DNA) as containing the fundamental information necessary for the duplication of the cell, for its growth, development, and function. Select portions of instructions are carried from DNA to the ribosomal assembly line by another nucleic acid, messenger ribonucleic acid (mRNA), at which point it meets with still another type of nucleic acid, transfer (tRNA), each carrying a definite amino acid. Protein synthesis occurs at this juncture by a sequence of recogni tion of m- and tRNA, alignment, and formation of peptide bonds. The various enzymes and other proteins so pro duced in turn control the biochemical behavior, morpho logical form, and physiological function of the cell. The effect of altering a functional protein by carcinogen is re flected at the level of transcription of information from DNA or from mRNA. Pitot, of the University of Wiscon sin, believes that the stability of the messenger template 140
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could be greatly influenced by a carcinogen and manifest itself by conversion of a normal cell to a cancer cell. In view of the permanent and heritable alteration in the characteristics of neoplasms, research workers believe that the reaction of the carcinogen with DNA or RNA is more satisfying ideologically than the indirect effect with protein. Thus, they have given serious attention to demon strating experimentally that carcinogens combine with nu cleic acids. By means of highly sensitive procedures this combination has now been shown with a carcinogenic aro matic amine and an azo dye. But the evidence is best with nitrosodimethylamine and with some carcinogenic alkylat ing agents. Farber (University of Pittsburgh), Hultin (Wenner-Grens Institute, Stockholm), and Magee (MRC Toxicology Labs, England) have isolated 7-methylguanine after hydrolysis of RNA from the liver of rats treated with carbon-14-iabeled N-nitrosodimethylamine. 7-Methylguanine does not ap pear in normal RNA, although it has been found in the urine of control animals. Brookes and Lawley, and Haddow, at the Chester Beatty Institute in London, Boutwell at the University of Wisconsin, and Abell at the National Cancer Institute, Bethesda, have evidence that sulfur mus tard (yperite), other alkylating agents of the mustard type, and β-propiolactone also react with nucleic acids at specific sites. The significance of such interactions on the expression of information on the gene is being studied. In addition to a firm covalent binding (a temporary inclusion of the car cinogen in the DNA or RNA strands) a sort of clathrate complex (intercalation) has been proposed, principally to account for the carcinogenicity of relatively sluggishly re acting hydrocarbons. Research workers consider that the resulting deformation is sufficient to give altered mRNA upon transcription. Using sensitive techniques, investigators have shown that the chemical carcinogens interact with a number of crucial cell components. Future work will have to show, how ever, whether these reactions are related to the carcino genic process, or whether they simply reflect an alteration leading, perhaps, to death of the cell. Considering the ad vances being made in fundamental biochemistry relating to events surrounding cell growth, multiplication, function, and differentiation and the awareness of many investigators of the implication of their findings in respect to abnormal processes such as cancer, research workers hope to develop insight into the crucial initiation reaction. Growth of Cancer Cells Normal and cancerous tissues result from a multiplica tion of individual cells. The conditions controlling develop ment may be quite different from the formation of the initial tumor cell. Some intriguing experiments demon strate this point. For example, more than 20 years ago Tannenbaum at Michael Reese Hospital, Chicago, showed that tumor development in mice which had been given identical preliminary treatment with a carcinogen depended greatly on dietary conditions. A restricted diet gave a lower tumor incidence in animals than an unrestricted diet. In our discussions on azo dye and ZV-2-fluorenylacetamide carcinogenesis, we mentioned the hormonal modification of the carcinogenic process. A rat in which the pituitary gland (hypophysis) has been removed does not develop liver tumors when fed these carcinogens. However, the
livers of hypophysectomized animals do contain almost as much "protein-bound carcinogen" as their normal controls. This equality may mean that initiation has occurred bet that the development of the tumor cells requires a hormonal stimulation absent in hypophysectomized animals. We have been able to demonstrate the reverse relationship. In the presence, of an excess of hormones, liver carcinogenesis proceeds more rapidly than in the controls. Similar findings have been made with other carcinogens and with other organs that are responsive to hormones. For example, mammary tumors in rats can be induced more rapidly by the simultaneous administration of carcinogen and pituitary hormones (also of drugs affecting the pituitary-hypothalamus-endocrine axis). The host supplies growth-promoting agents which appear to favor the development of cancers at certain sites. There is a question whether the "promotion phase" operates for all cancers, but such conditions have not been investigated for all organs affected by chemical carcinogens. What is missing is knowledge of the exact nature of the factors provided by the host which apply to the development of cancers in a nonendocrine target. Active research in this area will help delineate the environment favoring or hindering the development of tumors. In addition to endogenous growth-promoting factors such as hormones, the immune status of the host plays a role in the eventual outcome of the carcinogenic process. Early cancer cells are different enough from normal to be recognized by immune defense systems, just as cells invaded by bacteria or viruses can be distinguished from normal cells. Cancer cells are, however, but weakly antigenic in most cases. Only recently were sensitive techniques developed that can detect antigenicity in chemically induced cancers. Removal of the thymus at an early age, which reduces the immunological competence of animals, as do other treatments (x-ray, cortisone), favors the development of chemically induced cancers. Such a relationship is further indirect evidence of the antigenic activity of chemically
induced cancer. On the other hand, potentiation of immune response reduces the incidence of tumors and is one additional novel area by which cancer induction can be influenced. Currently there is also interest in a viral etiology for cancer, because a few types of cancer in animals are caused by specific viruses. Certain noncarcinogenic viruses can potentiate the action of a chemical carcinogen. Some chemical carcinogens depress interferon production, which in turn might cause proliferation of a repressed viral agent. In the context of the types of chemical carcinogens surveyed in this article, however, it is farfetched to implicate an indirect, virus-mediated mechanism. The growth of tumors is significantly modified by a variety of host factors. Differences in cancer induction in various species (that is the outstanding sensitivity of some animals and the unusual resistance of others to identical chemical treatment) may be based on endogenous conditions. Study of such differences is of general interest since the mechanisms of growth, differentiation, and development hinge on understanding the role of hormonal, immunological, and genetic factors, complex and intriguing fields. Understanding of this phase of the carcinogenic process will help investigators to clarify it. And it could provide important implications for prevention, or at least moderation, of the process.
Cancer can spread as metastases from one tissue to another within the body. In the photomicrographs shown here, the
malignant cells of the uterus (left) have metastasized to form a cancer in the kidneys (right)
Progression of Tumor
Cells
Progression does not apply to all neoplasms. Perhaps because of their growth characteristics or other special properties the early tumors are sometimes rather easily altered to a more malignant type. During this process, the functions of the normal cell which still may have been maintained in the initial tumor cell are generally lost. This phase of the cancer induction process, although quite important, is not yet very well developed from a chemical viewpoint and thus is far outside of the framework of this article.
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In respect to the principal carcinogenic agents discussed in this article, namely aromatic amines, azo dyes, and nitrosodialkylamines, certain general concepts can be de rived from the results of animal tests and possibly extrap olated to as yet untested structures. • Aromatic Amines: There is good evidence that an aromatic amine is carcinogenic when the amino group is located in such a position in a polynuclear aromatic ring that it is equivalent to the 2-position in naphthalene or in a para position to a biphenyl link. Compounds with an amino group in the 1-position of naphthalene or ortho to a biphenyl link very likely are ineffective. Compounds with —NH2 in a position meta to the biphenyl link are probably weakly active. Increases in carcinogenicity often occur when methyl or methoxy groups are substituted in the ring, particu larly para or ortho to the amino group. Substitution of fluorine or chlorine which does not increase the thickness of an otherwise planar molecule often enhances carcino genicity, especially in the ortho or para positions normally subject to biochemical detoxification reactions. Halogen atoms of larger size, bromine and iodine, have on occasion decreased the carcinogenic effect, but additional data must be secured. Substituents such as - O H , - C O O H , - S 0 3 H , which increase the polarity of the molecule, often lower carcino genicity. A few exceptions to this rule are known, es pecially for large molecules with attached polar groups. Consideration must be given not only to the substit uents themselves but to their possible biochemical trans formation products. Thus, methyl at certain positions is readily oxidized in vivo to carboxyl, leading to a less active product. On the other hand, relatively polar nitro deriva tives are readily reduced in vivo to the active hydroxylamino or amino derivatives.
SELECTED REFERENCES FOR ADDITIONAL READING
1. Arcos, J. C , Arcos, Martha, "Progress in Drug Re search," Fortschr. Arzneimittelforsch., 4: 408-^581, 1962. 2. Boyland, E., "The Biochemistry of Bladder Cancer," Springfield, 111., Charles C Thomas, 1963. 3. Carcinogenesis Studies Branch, Carcinogenesis Ab stracts, 3, 1965, National Cancer Institute, Bethesda, Md. 20014. 4. Clayson, D. B., "Chemical Carcinogenesis," Boston, Little, Brown & Co., 1962. 5. Eckardt, R. E., "Industrial Carcinogens," New York, Grune and Stratton, 1959. 6. Haddow, Α., Weinhouse, S., Adv. Cancer Res., 9, 1965 (a continuing series). 7. Homburger, F., Progr. Exptl. Tumor Res., 7, 1965 ( a continuing series ). 8. Scott, T. S., "Carcinogenic and Chronic Toxic Hazards of Aromatic Amines," New York, Elsevier Publishing Co., 1962. 9. Shimkin, M. B., "Science and Cancer," Washington, D.C., PHS Public. No. 1162, Superintendent of Docu ments, 1964. 10. Temkin, I. S., "Industrial Bladder Carcinogenesis," New York, Pergamon Press, 1963. 142
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• Azo Dyes: The general rules for the aromatic amines also apply to azo dyes. Additionally, however, there are substituents that hinder biochemical reduction of the azo link, especially if they are located in an appropriate posi tion on the ring, and, therefore, give a more active dye. In contrast, those substituents that enhance the reduction of the azo link with production of harmless, or at least less potent intermediates, lead to a decrease in the effect. •Nitrosodialkylamines: These compounds are active when the substitution on the amine includes residues which are susceptible to biochemical oxidation with con sequent formation of diazoalkane or carbonium ion inter mediates which have been postulated as the actual carcino gens. This area is a relatively novel one and for this reason will require additional experimental development. In any case, it is apparent from the discussion of the various structures which are carcinogenic that cancer can be elicited by a great diversity of chemical structures. Some of them are synthetic curiosities, others unfortunately are important in commerce, and others are not even manmade but are products of nature. Because of historical developments great emphasis is placed on the detection of the classic carcinogenic polynuclear aromatic hydro carbons such as benzopyrene or methylcholanthrene in the examination of suspect mixtures such as tobacco smoke and air and water pollutants. The presence of these hydrocarbons may actually help to explain the increased occurrence of cancer in certain populations, but it is also necessary to keep in mind that causes of cancer are not uniquely related to their presence. Thus, the habit of attempting to explain cancer hazards in terms of the presence of hydrocarbons should be thoroughly revised. The problem of cancer induction by environmental agents may be approached more successfully experimentally if an objective and broad-minded attitude is assumed.
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