N-Nitroso Compounds: Diet and Cancer Trends - ACS Symposium

21 N-Nitroso Compounds: Diet and Cancer Trends

Downloaded by UNIV OF CALIFORNIA SANTA BARBARA on June 10, 2016 | http://pubs.acs.org Publication Date: December 9, 1981 | doi: 10.1021/bk-1981-0174.ch021

An Approach to the Prevention of Gastric Cancer JOHN H. WEISBURGER American Health Foundation, Valhalla, NY 10595

Sensitive analytical techniques can detect nitros amines in the environment. Thus far no human cases of cancer attributable unambiguously to short chain dialkylnitrosamines detected in foods have been ob served in the Western world. Burnt and unburnt to bacco contains appreciable amounts of nitrosonor nicotine and related compounds that have been in criminated in tobacco carcinogenesis. Certain ni trosamines were identified in foods consumed in China in areas with a high incidence of esophageal cancer and a cause-effect relationship has been suggested but not yet proven. Eastern Iran and Southern regions of the U.S.S.R. also have a high incidence of esophageal cancer but without such d i rect evidence of dietary nitrosamines. Nonethe less, a l l of the high risk regions have in common low intakes of foods with vitamin C. Gastric can cer can be induced in animals by alkylnitrosoureido compounds. These can form from nitrite and suit able substrates, a reaction that can be inhibited by vitamins C and Ε. The risk factors for human gastric cancer include foods pickled with salt and saltpeter or residence in areas with high geochemical nitrate. We have found mutagenic activity for Salmonella typhimurium TA 1535 with an extract of a pickled fish frequently eaten in high risk Japan, which also induced glandular stomach cancer in rats. The formation of the mutagen was inhibited by vitamin C. Thus, gastric cancer may stem from consumption of specific pickled foods or residence in areas with high soil nitrate without the simul taneous consumption of foods containing sufficient inhibitory vitamins C or E. This concept accounts for the pronounced decrease in stomach cancer in the United States. 0097-6156/81/0174-0305$05.00/0 © 1981 A m e r i c a n Chemical Society Scanlan and Tannenbaum; N-Nitroso Compounds ACS Symposium Series; American Chemical Society: Washington, DC, 1981.

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Are Nitrosamines Human Carcinogens? The effect of the prototype dimethylnitrosamine or N-nitrosodimethylamine as a carcinogen was discovered about 1955 when Barnes and Magee, then at the Toxicology Center of the Medical Research Council of Great B r i t a i n , were asked to investigate i n animal models a finding of severe l i v e r t o x i c i t y i n three men i n a laboratory accident involving this chemical. They found that dimethylnitrosamine was indeed severely hepatotoxic (1_,2)· As an extension of this research, it was discovered that this chemical was also a powerful carcinogen for the l i v e r and secondarily for the kidney of rats. In the next 25 years, nitrosamines as a class were found to be potent carcinogens, the specific action of which depended on their structure; frequency and route of admini stration; species, s t r a i n , and other variables. These carcino gens permitted the induction of cancer at many target sites as a function of structure and metabolism (_1,.3,4). Just as their chemistry would predict, it was also found that nitrosamines could be formed from n i t r i t e and the appropri ate amines or amides at the pH found i n the stomach (5). Even t e r t i a r y amines could interact with n i t r i t e and form d i a l k y l n i trosamines (6, 7). In addition, the analytical chemistry used to detect nitrosamines has improved importantly. Whereas 25 years ago techniques were suitable only to detect parts per thousand, at this time, through instruments such as the Thermal Energy Ana lyzer designed s p e c i f i c a l l y to detect nitrosamines, levels of parts per b i l l i o n are easily quantitated, and even parts per t r i l l i o n can be detected (8)· To be t o t a l l y r e l i a b l e , however, such measurements need to be confirmed by other analytical sys tems including mass spectroscopy. Occasionally a r t i f a c t s seem to activate the s p e c i f i c instrumentation used for nitrosamines. In any case, these very sensitive analytical systems have now demon strated the occurrence of nitrosamines at low but definite levels i n the human environment. I t i s probable, although not yet so demonstrated, that 100 years ago, when saltpeter was used exten sively i n the preservation and pickling of many kinds of foods (9), that the levels of nitrosamines i n the human environment were quite appreciable. Yet, except f o r specific cases of acute poisoning (2,10, 11), and thus mostly of l i v e r damage, there appears to be no r e l i a b l y documented evidence that there are any cases of human can cer which can be unambiguously associated with an antecedent ex posure to nitrosamines i n the Western world (12). Naturally Occurring or Endogenously Produced Nitrosamines as Human Carcinogens. On the other hand, as discussed at this meeting by Hoffmann et a l . (13,14), there are certain tobacco-specific nitrosamines such as nitrosonornicotine which form a substantial part of the

Scanlan and Tannenbaum; N-Nitroso Compounds ACS Symposium Series; American Chemical Society: Washington, DC, 1981.


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genotoxic carcinogens i n tobacco smoke and i n tobacco chews. Specific association of these kinds of carcinogens with cancer at sites related to the use of tobacco, such as cancer of the lung, pancreas, kidney, urinary bladder, and also, i n the presence of heavy alcohol intake, cancers of the oral cavity and esophagus remains to be f u l l y documented (15-22)· I t i s reasonable to as sume, however, that these carcinogens play a role f o r cancer i n these target organs that accounts for a substantial part of the cancers i n various countries* In addition, i n certain parts of the world, such as Eastern Iran, Southern Soviet Union, and Central China, cancer of the esophagus i s seen i n people who do not appear to smoke and drink heavily. Despite extensive cooperative efforts between i n v e s t i gators i n Iran and the International Agency for Research on Can cer, the etiology of esophageal cancer i n Iran remains obscure (23, 24, 25). Nitrosamines have been suspected as etiologic fac tors but have not been found as yet. Nonetheless, the n u t r i t i o n a l intake i s poor i n green and yellow vegetables, f r u i t s , and other vitamin C and Ε containing antidotes to n i t r i t e (25), and thus, through this association, the endogenous formation of n i trosamines cannot be ruled out. This i s especially true inasmuch as recent data from China (26-29), the easternmost extension of the belt of esophageal cancer incidence, does suggest that n i t r o samines are present i n that environment. Evidence That Nitrosamides May Be Human Carcinogens. While attempting to examine the question whether some power f u l bacterial mutagens, i n this instance N'-methyl-N'-nitro-N-nitrosoguanidine (MNNG), were carcinogenic, Sugimura and Kawachi (30) administered this mutagen, a water-soluble, rather polar substance, to rats i n drinking water. The important discovery was made that this mutagen not only was a powerful carcinogen but r e l i a b l y induced cancer i n the glandular stomach, mimicking accu rately the disease seen i n man. A detailed study of the patho genesis of this then new animal model revealed that this chemical taken orally not only induced the neoplasm but also induced a l l the antecedent and accompanying lesions such as g a s t r i t i s and i r i tes t i n a l i z a t ion of the gastric epithelium, as seen i n man (30, 31, 32). Salt exerts a promoting effect (33). The effect of salt as a promoter i s linked to the epidemiologic information that popu lations with a high incidence of gastric cancer also tend to ex h i b i t a high incidence of hypertension (34-40). We have linked the gastric cancer model of Sugimura to the examination of the occurrence of human gastric cancer and to con sideration of the underlying mechanisms.


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Nitrosamides and Gastric Cancer» Between 1900 and 1950, gastric cancer was one of the major types of cancer i n the United States. I t s incidence and mortal i t y have decreased sharply since 1950 (41), and the concepts pre sented i n this paper account for this decline. In other parts of the world, including Japan, the mountainous i n t e r i o r regions of Central and Western Latin America, and i n Northern and Eastern Europe, as well as i n Iceland, gastric cancer remains one of the major cancers. There i s a North-South gradient, or South-North i n the Southern hemisphere, with greater incidence i n more f r i g i d zones. From geographic pathology and migrant studies, stems the view that n u t r i t i o n and specific dietary factors were associated with gastric cancer (34,42,43,44). Typically, high r i s k groups eat diets r i c h i n carbohydrates, without any direct role i n the etiology, but with the significant inclusion of dried and salted f i s h or certain pickled or smoked foods. Also, there was a v a r i able intake of fresh f r u i t s and vegetables, namely, a seasonal low consumption of such foods during winter and spring. The geo chemistry often involved elevated s o i l nitrate and, thus, foods and water r i c h i n nitrate (45,46,47). We have suggested that an alkylnitrosoureido type of com pound, such as the one, mentioned above, developed by Sugimura to induce glandular gastric cancer i n animal models, may be involved i n human gastric carcinogenesis (48). The required n i t r i t e might be derived from: (1) i t s reductive formation i n foods r i c h i n nitrate and stored at room temperature after cooking, (2) the consumption of pickled foods, and (3) consumption of smoked food, i n some parts of the world. I t has also been observed that s i z able amounts of n i t r i t e are produced by the reduction of nitrate secreted by the salivary glands through the oral microbiologic f l o r a . The nitrate stems from the oral intake of foods r i c h i n this compound either through s o i l chemistry or by addition i n the p i c k l i n g process (49-52). Precursors such as amides can be con verted by n i t r i t e to carcinogenic nitrosamides i n the acidic en vironment of the stomach or during pickling i n the presence of acids such as acetic acid (vinegar) or l a c t i c acid (5). Mirvish (53,54) discovered that vitamin C could i n h i b i t n i trosation reactions. The purely chemical interaction of ascorbic acid with n i t r i t e has been studied for theoretical reasons and because of i t s importance i n the preservation of foods. This i n teraction has received increased attention for minimizing the presence of nitrosamines and nitrosamides i n the environment, and especially i n foods. We have studied the relationship i n gastric carcinogenesis between high levels of n i t r i t e , including pick l i n g , and of vitamin C as a protective and i n h i b i t i n g element. Vitamin Ε can also i n h i b i t nitrosation reactions but the mechanisms may be somewhat different than those for vitamin C (55,56). Of course, vitamin C i s water soluble while vitamin Ε



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i s l i p i d soluble. Certain nitrosated substrates are l i p i d solu ble, and thus vitamin Ε may offer certain advantages as an i n hibitor. We used an experimental model, the f i s h Sanma, which i s eat en i n a region of Japan at high r i s k for gastric cancer. Treat ment of homogenates of this f i s h with n i t r i t e at pH 3 led to the development of a direct-acting mutagen for Salmonella typhimurium TA 100. The formation of mutagens could be completely blocked by vitamin C (57). This property i s similar to the i n h i b i t i o n of the formation of nitrosamines and nitrosamides, discussed above, and i s compatible with the view that the mutagens from f i s h are compounds with a nitrosamide type of functional group. Sanma yielded more mutagenic a c t i v i t y than other types of f i s h , but it i s important that several types of meat f a i l e d to produce such direct-acting mutagens (57). We demonstrated that the mutagenic a c t i v i t y from the reac tion of n i t r i t e and Sanma was carcinogenic (48). I t i s quite relevant that this product induced glandular stomach cancer i n rats. Since the formation of the mutagen can be blocked by v i t a min C, it would seem that the formation of glandular stomach can cer can also be so inhibited. Materials and Methods Treatment of Fish Extracts and Assays. The preparation of the f i s h extract has been described (57). B r i e f l y , the f i s h were homogenized and incubated with n i t r i t e (5 g of homogenate plus 5,000 ppm NaCl and 5,000 ppm NaN0 ) at pH 3 and 25°C for 3 hours. These levels were used to mimic the h i s t o r i c a l l y employed doses of saltpeter and salt i n pickling (9). Moreover, nitrosa t i o n can occur at lower levels of n i t r i t e (see Mirvish, p.271, i n ref. 8). The reaction was stopped by the addition of 5,000 ppm ammonium sulfamate. A control sample was treated likewise but without adding n i t r i t e . The mixture was extracted twice with hexane (removal of l i p i d fraction, which was mutagen-free) and four times with ether. The combined ether extracts were taken to dryness. The residue was redissolved i n water (adjusted to pH 3). The mutagenic a c t i v i t y of this solution was determined by conventional Ames assay and the volume was adjusted so that 10 u l would y i e l d 125 His+ revertants/plate (spot t e s t ) . The extracts were prepared a week before use and were deep-frozen. The muta genic a c t i v i t y of the extracts was stable during storage f o r 3 weeks· The mutagenic a c t i v i t y of the extracts was assayed using Salmonella typhimurium s t r a i n TA 1535, as described by McCann et a l . (58). Ten u l of f i s h extract were spot-tested without addi t i o n of an S9-enzyme preparation (57). MNNG (20 ug/plate) was used as a positive control. Bioassays— Male random-bred Wistar rats, 50 days old, on lab chow and water ad libitum, were divided into two groups. The 2



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control group (I) was given extracts from f i s h homogenates not treated with n i t r i t e three times per week for s i x months by ga vage. The experimental group ( I I ) received the extract from f i s h treated with n i t r i t e . The weekly amount of extract given was calculated to have mutagenic a c t i v i t y (Ames assay) equal to a weekly oral dose of MNNG active i n inducing adenocarcinomas of the glandular stomach i n rats (59). Thus, the rats received 0.5 ml per gavage of the extract during the f i r s t two months of the treatment period and 1.0 ml per gavage of extract during the re maining four months. A rat received per week the extract from about 100 g ( i n i t i a l two months of treatment period) or about 200 g (remaining four months of treatment period) of f i s h . The a n i mals were held for an additional observation period of 18 months. The surviving rats were k i l l e d and a l l animals were carefully autopsied with emphasis on the gastrointestinal tract. The tissues were fixed and processed by conventional histologic techniques and studied microscopically. Results Two of the ten rats from Group I died from causes unrelated to treatment, mainly pneumonia or other infectious processes. Three rats died after l i v i n g more than 18 months. Five rats were a l i v e at the 24-month point. In Group I I , three of the rats giv en the extract of n i t r i t e - t r e a t e d f i s h died early, due to causes unrelated to treatment. Five rats died after 18 months and their tissues were available for study. F i n a l l y , seven rats a l i v e at 24 months were k i l l e d . Thus, there was a t o t a l of 8 rats for evaluation i n the con t r o l group and 12 i n the experimental group. In the control group given an extract of f i s h alone, there were no neoplastic changes i n the stomach or pancreas except an e p i t h e l i a l hyper plasia of the glandular stomach of one rat (Table 1). Instead, there were tumors i n the testes, kidneys, and soft tissues, as are often seen i n aged rats. In the group given the extract of n i t r i t e - t r e a t e d f i s h , glandular hyperplasia of the stomach was seen i n s i x rats (Table 1). In most, there was also i n t e s t i n a l metaplasia. In addition, f i v e rats had squamous c e l l hyperplasia of the forestomach epi thelium. Five tumors, including two adenocarcinomas and one adenosquamous c e l l carcinoma, occurred i n the glandular stomach of the four animals of the experimental group (Table 2). One rat had a tubular adenoma and simultaneously a well differentiated tubular adenocarcinoma. Another histologic type of adenocarcin oma was a poorly differentiated carcinoma (scirrhous type). In the forestomach, squamous c e l l carcinoma developed i n two of these rats. Of interest also i s the finding of an adenocarcinoma i n the small intestine of one r a t , and pancreatic acinar c e l l tumors i n three animals, a l l of which may be s i g n i f i c a n t l y r e l a t ed to the treatment. In addition, there were several other types of neoplasms i n this group.



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Table I . Incidence of Stomach Epithelium Hyperplasia and I n t e s t i n a l Metaplasia (66) Experimental Group and Treatment

Effective Forestomach Number of Squamous C e l l Rats Hyperplasia

1

8 Fish extract alone II Fish extract + NaN0

12

Glandular Stomach Glandular Intestinal Hyperplasia Metaplasia

0

l

5*>

6C

a

1

6

N-Nitroso Compounds: Diet and Cancer Trends - ACS Symposium

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