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Diet, Nutrition, and Cancer (1982)

Chapter: 13 Mutagens in Food

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Suggested Citation:"13 Mutagens in Food." National Research Council. 1982. Diet, Nutrition, and Cancer. Washington, DC: The National Academies Press. doi: 10.17226/371.
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13 Mutagensin Food As interest in the possible relationship between diet and cancer has increased in recent years, so have attempts to determine whether chemical carcinogens may be present in our foods. The foods that we eat contain a vast number of separate chemical entities: several thousand as additives and many times this number as natural constitu- ents. Of course, most of these chemicals are present in relatively low concentrations, but if potent carcinogens exist, even at low con- centrations in commonly consumed foods, they may warrant concern. The problem, therefore, is how to test the very large number of chemicals present in the complex mixtures we call food to determine whether or not they may be contributing to our risk for cancer. An adequately performed chronic feeding study in rodents to determine whether a chemical is a carcinogen takes several years to complete and analyze and can cost more than $500,000. Therefore, the use of simpler and less expensive tests may be considered, at least to help us determine which chemicals to subject to long-term studies. As discussed elsewhere in this report, initiation of the carcino- genic process may involve an alteration in the genetic material of a cell. Therefore, it is reasonable to suppose that chemicals that alter DNA (e.g., cause mutations) will have a high probability of being initiators of carcinogenesis. The fact that DNA is chemically similar in all living organisms means that even chemicals that cause mutations in bacteria can be suspected as potential carcinogens in humans. In several extensive studies conducted in independent lab- oratories, the correlations between mutagenic activity in bacteria and carcinogenicity in mammals have been analyzed (McCann and Ames, 1976; McCann et al., 1975; Purchase et al., 1978; Simmon, 1979; Sugimura et al., 1976~. It is clear from these and other studies that a chemical found to be mutagenic in any living system should be suspected of being carcinogenic. However, it is impossible to provide a single number to express the degree of confidence with which a mutagen can be considered to be a carcinogen or with which a nonmutagen can be regarded as a noncarcinogen. This uncertainty arises from several sources, the most important of which is that the correlation between mutagenicity and carcinogenicity is highly de- pendent upon the class of chemical being investigated. For some classes of chemical carcinogens, such as aromatic amines, polycyclic hydrocarbons, and direct alkylating agents, there appears to be a high degree of correlation. However, it is difficult to detect the mutagenic activity of some types of carcinogens, especially highly chlorinated compounds. Therefore, judgment must be exercised, in- cluding a careful consideration of the structure and likely metabo- lites of the chemical under test, when the significance of a positive or negative mutagenicity test is being evaluated. 13-1 277

278 DIET, NUTRITION,AND CANCER The utility of mutagenicity tests in identifying chemical carcino- gens and the subsequent removal of these compounds from products to which humans are exposed can be illustrated by several historical ex- amples. These would include the food preservatives 2-~2-furyl)-3- (5-nitrofuryl~acrylamide (AF-2), which was extensively used in Japan, the flame retardant chemical tris(2,3-dibromopropyl~phosphate, which was widely used in children's sleepwear in the United States, and the hair dye ingredient 2,4-diaminoanisole. The fact that simple muta- genicity tests correctly predicted the carcinogenic potential of these chemicals adds to our confidence that correctly interpreted muta- genicity data can assist us in identifying environmental carcinogens. The most widely used of the mutagenicity assays is the Salmonella plate incorporation test, commonly known as the Ames test. In this assay, a chemical is tested for its ability to induce mutations in different strains of a bacterium (Salmonella typhimurium). Most chemical carcinogens and mutagens do not interact directly with DNA. They require alteration by enzymes in order to become activated. This process of "metabolic activation" cannot usually be accomplished by enzymes present in bacteria. Therefore, in the Salmonella test, an extract of mammalian liver (usually from the rat) is added to provide the enzymes necessary for metabolic activation. Many mutagenic test systems other than S. typhimurium have been used to test chemicals (see review by Hollstein and McCann, 1979~. In the discussion that follows, however, most of the studies discussed involve S. typhimurium. Mutagenicity assays have also been used to investigate the interactions between chemicals. This has resulted in the discovery of both comutagens, which enhance mutagenic activity of other chemicals, and inhibitors of mutagenesis. The knowledge that a chemical is a comutagen or an inhibitor of mutagenesis can provide us with a useful tool for investigating the metabolic fate and genetic interactions of chemicals. Modification of mutagenic activity, par- ticularly as determined in _ vitro test systems, frequently has no relevance to _ viva effects. Specific in vitro effects of modifiers of mutagenesis, such as inhibition of a particular metabolizing enzyme, for example, may not operate or may even have the opposite effect in living organisms. However, where modification of mutagenesis is ob- served, the mechanism should be elucidated. MUTAGENS RESULTING FROM COOKING OF FOODS Benzo[~]pyrene and Other Polynuclear Aromatic Hydrocarbons Almost 20 years ago Lijinsky and Shubik (1964) and Seppilli and Sforzolini (1963) reported that beef grilled over a gas or charcoal fire contained a variety of polycyclic aromatic hydrocarbons (PAH's). Benzo~a~pyrene was found in charcoal-broiled steak in levels up to 8 g/kg (Lijinsky and Shubik, 1964~. The source of the PAM's resulting 13-2

Mutagens in Food! 279 from charcoal broiling was the smoke generated when pyrolyzed fat dripped from the meat onto the hot coals. Thus, meats with the highest fat content acquired the highest levels of these chemicals (Lijinsky and Ross, 1967~. When meat was cooked in a manner that prevented expo- sure to the smoke generated by the dripping fat, this source of contam- ination was either reduced or eliminated (Lijinsky and Ross, 1967; Lintas _ al., 1979; Masuda et al., 1966~._ _ PAM's have also been found in a variety of smoked foods and in roasted coffee (Howard and Fazio, 1980~. Vegetables can easily become contaminated by PAM's from air, soil, or water; fish and shellfish can assimilate such chemicals from their marine environments (Howard and Fazio, 1980~. However, unless vegetables or seafood are obtained from highly contaminated environments, the major source of PAR will probably be the smoking or cooking of food. Mutagens from Pyrolyzed Proteins and Amino Acids During the past few years, it has become clear that PAM's account for only a small fraction of the mutagenic (and, therefore, potentially carcinogenic) activity that occurs in foods during cooking. Nagao et al. (1977a) used dimethylsulfoxide to prepare extracts of the charred surfaces of broiled fish and meat. They found that the mutagenic acti- vities of these extracts for histidine-requiring strains of S. typhi- murium were hundreds or thousands of times greater than could be accounted for by the reported benzo~aipyrene contents of these cooked foods. For example, the mutagenic activity of charcoal-broiled beef- steak was the equivalent to that of approximately 4,500 fig of benzo~a]- pyrene per kilogram of steak, even though Lijinsky and Shubik (1964) had reported that charcoal-broiled steak contained no more than 8 fig of this chemical per kilogram. The mutagenic activity in the broiled fish and beef could also be detected in S. typhimurium strain TADS, implying that the agent could induce frameshift mutations (Nagao et al., 1977a; Sugimura et al., 1977~. Positive results in these assays depended on the presence of an _ vitro metabolic activation system utilizing the postmitochon- drial supernatant from homogenized livers of rats pretreated with polychlorinated biphenyls. Bjeldanes et al. (in press, a, b) have re- cently completed a series of detailed studies on the cooking condi- tions under which mutagenic activity is produced in various types of fish, meats (including organ meats), as well as eggs, milk, cheese, and tofu. To determine what constituent or constituents of fish and meat contribute to the mutagenic activity produced by cooking, studies have been conducted to examine the mutagenicity of smoke condensates from various substances. Smoke obtained from pyrolyzed proteins, such as lysozyme and histone, was found to be highly mutagenic to S. typhi- murium, whereas smoke condensates from pyrolyzed DNA, RNA, starch, or vegetable oil were only slightly mutagenic (Nagao et al., 1977b). 13-3

280 DIET, NUTRITION, AND CANCER Pyrolysis of tryptophan resulted in more mutagenic activity than did any other common amino acid, but almost all of the amino acids tested yielded some mutagenic activity when pyrolyzed (Matsumoto et al., 1977; Nagao et al., 1977c). Purification of the mutagenic products resulting from pyrolysis of tryptophan resulted in the isolation of two previously unknown amino-Y- carbolines that are potent mutagens: 3-amino-1,4,-dimethyl-5H-pyrido- [4,3-b~indole (referred to as Trp-P-l, for "Tryptophan Pyrolysate 1") and 3-amino-1-methyl-5H-pyrido[4,3-b~indole (Trp-P-2) (Akimoto et al., 1977; Sugimura et al., 1977; Takeda et al., 1977~. The mutagenic activity resulting from pyrolysis of L-glutamic acid was shown to be due to the formation of 2-amino-6-methyldipyrido- [1,2-a:3'2'-diimidazole (Glu-P-l) and 2-aminodipyrido[1,2-a:3',2'-d]- imidazole (Glu-P-2) (Yamamoto et al., 1978~. The structural simi- larity between these products of glutamic acid pyrolysate and Trp-P-1 and Trp-P-2 is evident from Figure 13-1. Wakabayashi et al. (1978) isolated a different, but structurally related, heterocyclic mutagen from pyrolyzed lysine. This compound was 3,4-cyclopentenopyrido[3,2-aicarbazole (Lys-P-l). Pyrolysis of phenylalanine resulted in the formation of the mutagen 2-amino-5- phenylpyridine (Phe-P-l) (Sugimura et al., 1977~. When soybean globulin was pyrolyzed, the substances that contrib- uted to the mutagenic activity were compounds not previously identified as pyrolysis products of any individual amino acid. These compounds, 2-amino-9H-pyrido[2,3-b~indole (A~C) and 2-amino-3-methyl-9H-pyrido- [2,3-b~indole (Medic), are quite closely related to the Y-carboline compounds Trp-P-1 and Trp-P-2 (Yoshida et al., 1978~. Uyeta _ al. (1979) found that both Trp-P-1 and Trp-P-2 were present in pyrolysates of casein and gluten. Yamaguchi et al. (1979) identified Glu-P-2 in the tar resulting from pyrolysis of casein. These investigators estimated that Glu-P-2 and Glu-P-1 accounted for approximately 10% of the total mutagenic activity of the pyrolysate. Analyses have confirmed that at least some of the mutagenic pyrolysis products of amino acids are present in cooked foods. For example, Trp-P-1 has been found in "very well done" broiled beef and Glu-P-2 in broiled cuttlefish, although they account for less than 10% of the total mutagenic activity in extracts of these foods (Yamaguchi et al., 1980a,b). Similarly, sardines broiled to a dark brown color contain Trp-P-l, Trp-P-2, and Phe-P-l, although most of the mutagenic activity in these fish was due to the presence of other compounds (Yamaizumi et al., 1980) (Table 13-1~. Pieces of beef or chicken grilled in a high gas flame contained A~C and MeA~C (Matsumoto et al., 1981~. Similarly, A~C could be identified in grilled onions. 13-4

Mutagens in Food 2X1 From Amino Acids: CH3 NH2 HCH3 Trp-P-1 4NH2 CH3 Glu-P-1 Lysine pyrolysate NJ0N W H Lys-P-1 From Proteins: N>lN~ NH2 H ARC Tryptophan pyrolysates Glutamic acid pyrolysates Soybean globulin pyrolysates CH3 ~NH2 H Trp-P 2 O ~r NH2 Glu-P-2 Phenylalanine pyrolysate JJ tN1NH2 Phe-P-1 ~N~N4NH2 H MeAaC FIGURE 13-1. Some mutagens from pyrolysates and from cooked foods. (Figure continued on next page.) 13-5

282 DIET, NUTRITION, AND CANCER Figure 13-1 (continued): Some mutagens from pyrolysates and from cooked foods. From Broiled Sardines: N=( 1 / N CH3 NH2 IQ From Broiled Beef: Protein pyrolysate NH2 N=< H3C~,N`~N CH3 MeIQx Protein pyrolysates NH2 NJ4` cH3 MeIQ Two previously unknown mutagens were isolated from broiled sardines (Kasai et al., 1979~. These were 2-amino-3-methylimidazo[4,5-fiquino- line (IQ) and 2-amino-3,4-dimethyllmidazo[4,5-fjquinoline (MeIQj, which are extraordinarily potent mutagens to S. typh~murium strain TA98 (Kasai _ al., 1980a,b,c). Except for the beef that contained IQ and possibly that containing MeIQ and 2-amino-3,8-dimethylimidazo-[4,5-f]- quinoxaline (MeIQx), the foods listed as sources of mutagens in Table 13-1 appear to have been very well cooked and even charred on the surfaces to produce the mutagenic compounds identified in the table. Information on mutagens formed by cooking foods at lower temperatures is discussed in the next section. As discussed in Chapter 3, the mutagenic activity of a chemical in bacteria indicates potential genotoxicity and possible carcinogenicity in mammals. To test for carcinogenic activity, it is necessary to use mammalian cell systems and intact mammals. Whenever other test systems also indicate genotoxic activity, it is more likely that a bacterial mutagen can act as a carcinogen. 13-6

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284 DIET, NUTRITION, AND CANCER Four of the mutagenic pyrolysates derived from amino acids or protein--Trp-P-l, Trp-P-2, Glu-P-l, and AolC--have been shown to in- duce sister chromatic exchanges in a permanent line of human lympho- blastoid cells (Tohda et al., 1980~. In addition, the basic fraction extracted from pyrolyzed tryptophan was found to cause mutations re- sulting in resistance to ouabain or 8-azaguanine in cultured Chinese hamster lung cells (Inui et al., 1980~. Trp-P-l, Trp-P-2, and Glu- P-1 can transform primary Syrian golden hamster embryo cells (Takayama _ al., 1977, 1979~. The cells transformed by Trp-P-2 have been shown to grow in soft agar and to result in tumors when inoculated into the cheek pouches of young hamsters with unimpaired immunocompetence (Taka- yama et al., 1978~. Although these findings support the potential car- cinogenicity of these chemicals, a definitive determination of carcino- genicity must be made in whole animals. Several of the mutagenic pyrolysates of amino acids or proteins have been tested for carcinogenicity in viva. Neoplastic nodules, which are presumed to be precancerous changes, were found in the livers of Wistar rats given the basic fraction from pyrolyzed trypto- phan at 0.2% in the diet (Matsukura et al., 1981b). Neither neoplas- tic nodules nor liver tumors had previously been observed in this strain of rats in this laboratory. Subcutaneous injection of Trp-P-1 (1.5 mg once a week for 20 weeks) induced sarcomas in Syrian golden hamsters and in Fischer rats (Ishikawa et al., 1979~. Trp-P-2 did not induce tumors in either hamsters or rats under the same experimental conditions (Ishikawa _ al., 1979~. Trp-P-1 and Trp-P-2 produced liver tumors in CDF1 (BALB/c x DBA) mice that were fed a diet con- taining 0.02% of either of these chemicals (Matsukura et al., 1981a). Some of these liver tumors metastasized to the lung. Female mice were more susceptible to these carcinogens than were the males. Six of nine female ACI rats fed 0.1% Trp-P-2 in their diet developed neoplastic nodules of the liver, and one of the six developed a hemangioendothe- lial sarcoma of the liver (Hosaka _ al., 1981~. None of the control animals developed such nodules or tumors. Glu-P-l, Glu-P-2, AaC, and MeArxC induced hepatomas in mice. Glu-P-1 and Glu-P-2 also induced hemangioendotheliomas between the scapulae of mice fed diets contain- ing 0.05% of either of these chemicals (Sugimura, in press). Thus, it appears that the identification of several of the mutagenic compounds found in pyrolysates of proteins and amino acids was an accurate pre- dictor of carcinogenicity. However, the presence of a carcinogenic chemical in a pyrolyzed amino acid or protein mixture does not neces- sarily imply that the carcinogen will also be present in normally cooked, uncharred food. Mutagens Fo~u~ed from Meat at Lower Temperatures In the experiments concerning the formation of mutagenic pyrolysis products from amino acids and proteins, temperatures of 250°C or greater were used (Matsumoto et al., 1977; 1978; Uyeta et al., 1979~. However, it is now known that simply boiling beef stock at temperatures 13-8

Mutagens in Food 285 of approximately 100°C results in the formation of bacterial mutagens (Commoner _ al., 1978; Vithayathil et al., 1978~. In fact, the forma-_ Lion of mutagens in beef stock has been detected at temperatures as low as 68°C (Dolara et al., 1979~. Frying of fish at 190°C produces mutagenic activity (Krone and Iwaoka, 1981~. Mutagenic activity also results when hamburgers are broiled, even when the surface temperature does not exceed 130°C (Weisburger and Spingarn, 1979~. A portion of the mutagenic activity formed from heated beef extract or from fried beef was found to be due to a chemical with a molecular weight of 198 (Spingarn et al., 1980a), which has now been shown to be IQ (Kasai et al., 1980a). MeIQx, another heterocyclic mutagenic compound that has not been identified as an amino acid or protein pyrolysate, has also been found in fried beef (Kasai et al., 1981~. However, the frying temperature was not specified. Weisburger and Spingarn (1979) suggested that this mutagen, formed in beef at moderate temperatures, may result from a browning reaction between sugars and amines rather than from the pyrolysis of proteins. Mutagen Formation Involving Carbohydrates If the formation of IQ during the cooking of beef results from a browning reaction, it might be expected that the browning of starchy foods could also result in the formation of mutagens. Spingarn et al. (1980b) have observed that the frying of potatoes and the toasting of bread result in the formation of mutagenic activity, but the chemi- cal~s) responsible for this activity and their source during the cooking process remain to be determined. Browning of foods results from the reaction of amines with sugars. Using a model system for the browning reaction, Spingarn and Garvie (1979) found that mutagenic activity occurred when any of six different sugars, including glucose, were refluxed with ammonium hydroxide. Sev- eral laboratories have found that heating a mixture of the amino acid lysine with glucose at temperatures between 100°C and 121°C results in products that are mutagenic (Powrie et al., 1981; Shinohara et al., 1980; Yoshida and Okamoto, 1980~. The increase in mutagenic activity with time paralleled the increase in browning (Shinohara et al., 1980~. Mutagenic activity could also be produced by using certain amino acids other than lysine (Powrie et al., 1981; Yoshida and Okamoto, 1980) or by using fructose rather than glucose (Powrie et al., 1981~. Chromosome aberrations are alterations in the structures of chromo- somes that can be observed through a microscope. Such aberrations are not likely to be heritable. The significance of their induction in cells _ vitro is not clear, particularly for chemicals unable to in- duce heritable mutations or _ vivo chromosome aberrations. Pyrazine and four of its alkyl derivatives--compounds formed by heating mixtures of sugars and amino acids (Koehler et al., 1969~-- were found to be nonmutagenic to S. typhimurium but capable of induc- ing chromosome aberrations in cultured Chinese hamster ovary (CHO) 13-9

286 DIET, NUTRITION, AND CANCER cells (Stich et al., 1980~. Commercial caramel and caramelized sam- ples of several sugars prepared by heating sugar solutions also caused chromosome aberrations in CHO cells (Stich et al., 1981b). Similarly, furan and six of its derivatives, which can be produced in foods by heating carbohydrates (Maya, 1979), were found to cause chromosome aberrations in CHO cells but to be nonmutagenic to bacteria (Stich et al., 1981a). PLANT FLAVONOIDS Among the most widespread of the known naturally occurring mutagens (possible carcinogens) that are normal constituents of many foods are the mutagenic flavonoids. Among the flavonol aglycones that have been shown to be mutagenic to S. typhimurium are quercetin, kaempferol, and galangin (Bjeldanes and Chang, 1977; Brown, 1980; Hardigree and Epler, 1978; Ma cGregor and Jurd, 1978). Quercetin has also been reported to induce gene conversion in yeast (Hardigree and Epler, 1978), transforma- tion of both hamster embryo cells (Umezawa et al., 1977) and BALB/c 3T3 mouse cells (Meltz and MacGregor, 1981), and mutations and single- stranded DNA breaks in L5178Y mouse cells (Meltz and MacGregor, 1981~. Both quercetin and kaempferol have been reported to cause mutations in V79 Chinese hamster cells (Maruta et al., 1979) and heritable mutations (sex-linked recessive lethals) in the fruit fly Drosophila melanogaster (Watson, 1982~. In some mutagenic plant products consumed by humans, the mutagenic substances isolated were identified as flavonoids. For example, most of the mutagenic activity of an acid hydrolysate of green tea could be accounted for by three flavonoids: kaempferol, quercetin, and myricetin (Uyeta et al., 1981~. The flavonoids kaempferol and isorha~nnetin were found to be responsible for most of the mutagenic activity found in Japa- nese pickles (Takahashi et al., 1979~. The mutagen in the spice of sumac was found to be quercetin (Seino et al., 1978~. Isorhamnetin and quercetin were the major mutagens in a methanol extract of dill weed (Fukouka et al., 1980~. Brown (1980) reported that the edible portions of most food plants contain flavonoid glycosides, especially quercetin and kaempferol. He estimated that the average daily intake of flavonoids in the U.S. diet is approximately 1 g and that the daily intake of mutagenic flavonoid glycosides may be equivalent to approximately 50 mg of quercetin. Approximately 25% of the flavonoid intake is derived from tea, coffee, cocoa, fruit jams, red wine, beer, and vinegar (Brown, 1980~. In view of the mutagenic activity and widespread distribution of certain flavonoids, particularly quercetin, it is important to deter- mine the carcinogenic potential of these chemicals. At present, the data concerning the carcinogenicity of quercetin are contradictory. Pamukcu et al. (1980) reported that adding 0.1% quercetin to the diet 13-10

Mutagens in Food 2SB7 of albino Norwegian rats for 58 weeks resulted in the induction of tumors in the epithelium of the intestine and urinary bladder. How- ever, when Saito et al. (1980) fed 2% quercetin to ddY mice throughout the lives of the animals, they found no significant increase in tumor incidence. At doses as high as 10% quercetin in the diet fed through- out life, no significant increase in tumor incidence was observed in ACT rats (Hirono et al., 1981) or in hamsters (Morino et al., 1982~. The reason for the discrepancy between the findings of Pamukcu et al. and the other investigators is not clear, but may relate to differences in sensitivity among the species and strain tested. Flavonols often exist in plants in the form of glycosides. For example, rutin is a glycoside of quercetin that can be hydrolyzed to release quercetin by enzymatic or chemical treatment. Such hydrolysis, mediated by intestinal bacteria, occurs when glycosides are consumed in foods. Rutin and other glycosides of mutagenic flavonoids have been shown to be mutagenic to S. typhimurium following treatment with glyco- sidase-containing extracts of the mold Aspergillus niger (Nagao et al., 1981), the snail Helix pomatia (Brown and Dietrich, 1979), rat cecal contents (Brown and Dietrich, 1979), or human feces (Tamura et al., 1980~. Mutagenic activity of rutin has also been reported in S. typhi- murium in the absence of glycosidase treatment, but only at doses higher than those required when such treatment is used (Hardigree and Epler, 1978). MUTAGENIC ACTIVITY IN EXTRACTS OF FOODS AND BEVERAGES Several food substances have been reported to contain mutagenic activity, although the specific chemicals responsible for this activity have not yet been identified. For example, coffee is mutagenic to Salmonella typhimurium strain TA100, whether it is brewed, instant, or decaffeinated (Aeschbacher and Wurzner, 1980; Aeschbacher et al., 1980; Nagao et al., 1979~. Although caffeine has been reported to be muta- genic to bacteria (Clarke and Wade, 1975; Demerec et al., 1948, 1951; Gezelius and Fries, 1952; Glass and Novick, 1959; Johnson and Bach, 1965; Kubitschek and Bendigkeit, 1958, 1964; Novick, 1956), it could not have been responsible for the mutagenicity of coffee observed in these reports, since decaffeinated coffee was as mutagenic as regular coffee and caffeine itself was not detected as a mutagen under the test conditions used (Aeschbacher et al., 1980; Nagao et al., 1979~. Studies examining the possible carcinogen~city of coffee are discussed in Chap- ter 12. Black tea, green tea, and roasted tea were mutagenic to S. typhi- murium strain TA100 in the absence of added enzymes (Nagao et al., 1979~. When extracts of Aspergillus niger or human feces containing enzymes capable of hydrolyzing glycosides were added, tea became muta- genic to S. typhimurium strain TA98 (Nagao et al., 1979; Tamura et al., 1980~. Acid hydrolysis of green or black tea also caused mutagenic 13-11

288 DIET, NUTRITION, AND CANCER activity to be released (Uyeta et al., 1981~. The flavonols quercetin, kaempferol, and myricetin have recently been shown to account for most of the mutagenic activity of an acid hydrolysate of green tea (Uyeta et al., 1981~. Grape juice was also found to be mutagenic to strain TA98, although only when tested with fecal extracts containing glycosidases (Tamura et al., 1980~. Mutagenic activity has also been detected in concentrates of 17 out of 27 commonly consumed Chinese alcoholic beverages, mostly fermented from rice, glutinous rice, and barley (Lee and Fong, 1979~. The muta- genic spirits were those that had been distilled only once or to which herbs or meat had been added. Evaporated residues from 12 out of 13 Japanese, Scotch, and North American whiskies were found to contain mutagenic activity (Nagao et al., 1981~. This activity did not require the addition of glycosidases or mammalian enzymes. It was mutagenic to _. typhimurium strain TA100, but not to TA98, indicating that it was inducing base-pair substitution rather than frameshift mutations. Some French brandies and apple brandies were also mutagenic when concentrated or fractionated (Loquet _ al., 1981; Nagao et al., 1981~._ _ Extracts of relatively few fruits, vegetables, and beverages are mutagenic. When Stoltz et al. (in press, a,b) fractionated extracts of 28 beverages and 40 fruits and vegetables, only 3 beverages and 5 fruits and vegetables showed reproducible mutagenic activity. The activity of three of the fruits (strawberries, raspberries, and peaches) was due to residues of the fungicide captan. Quercetin accounted for the mutagenicity of the remaining fruit and vegetable (raisins and onions) as well as two of the beverages (red wine and grape juice). The mutagen in coffee was not identified. This extensive survey of frac- tionated extracts of 68 foods did not reveal the presence of any muta- genic chemicals or foods that had not been previously reported. Simi- larly, Bjeldanes et al. (in press, a,b) found no significant mutagenic activity in extracts of eggs, milk, cheese, or tofu unless they had been cooked at high temperatures or to the point of darkening. Thus, with the exception of mutagens produced by cooking, it seems unlikely that large numbers of mutagens remain to be discovered in common foods. MODIFIERS OF MUTAGENIC ACTIVITY ~ : A number of substances in food have been reported either to enhance or to diminish the mutagenic activity of other substances. Most of these effects have been observed in In vitro test systems, and their relevance to effects in intact mammals is unknown. Modifiers of muta- genicity can be either comutagens or antimutagens. A comutagen is a substance that enhances the mutagenic activity of a chemical although it is not in itself mutagenic. This enhancement may take one of two forms: it may strengthen the mutagenic response of chemicals that are themselves mutagenic or may create a mutagenic response from nonmutagens. Similarly, an antimutagen is a substance that reduces or eliminates the mutagenic activity of a mutagen. 13-12

Mutagens ill Food 289 Harman and Norharman , Among the more interesting comutagens discovered in recent years are hanman (1 methyl-~-carboline) and norharman (~-carboline). Norharman is present in tobacco tar (Poindexter and Carpenter, 1962) as well as in toasted bread, broiled beef, and broiled sardines (Yasuda et al., 1978~. Harman is found in these same sources as well as in mushrooms and in Japanese sake (Takase and Murakami, 1966; Takeuchi et al., 1973~. These compounds were identified as comutagens when fractionation of pyrolyzed tryptophan resulted in a significant loss of mutagenic activity on S. typhimurium. Mixing the fraction containing barman and norharman with the fraction containing Trp-P-1 and Trp-P-2 restored the mutagenic activity of these mutagens (Nagao et al., 1977d). Norharman, which is at most marginally mutagenic, has been shown to be comutagenic when mixed with a variety of chemicals, including 4-dimethylaminoazobenzene, (Nagao et al., 1977d), aniline, o-toluidine (but not m- or p-toluidine) (Nagao et al., 1977e), nitrosodiphenylamine (Wakabayashi et al., 1981), 3-aminopyridine, 2-amino-3-methylpyridine (Sugimura et al., 1982), 2-acetylaminofluorene, 2-aminofluorene, and N-hydroxy-2-acetylamino- fluorene (Umezawa et al., 1978~. These chemicals all require an in vitro mammalian metabolic activation system for mutagenic activity. In addition, norharman is comutagenic with N-acetoxy-2-acetylaminofluorene in the absence of any metabolic activation system (Umezawa et al., 1978~. Aniline, which is nonmutagenic in the absence of norharman, was believed for many years to be noncarcinogenic. Its weak carcinogenic activity has only recently been demonstrated (National Cancer Institute, 1978~. Although the mutagenic activity of a number of chemical carcino- gens can only be observed in the presence of norharman, data are insuffi- cient to justify recommending the inclusion of norharman in routine screening. The possibility that many false positive results might be obtained with norharman has not yet been ruled out. The mechanism of the comutagenic action of harman and norharman is still unclear. Although these comutagens are generally believed to exert their activity by affecting the metabolic activation of the test compounds, they may act through other mechanisms as well. For example, the mutagenicity of N-acetoxy-2-acetylaminofluorene, which does not require activation, was enhanced by the addition of harman and/or nor- harman (Umezawa et al., 1978~. A number of substances in foods can reduce the activity of certain mutagens in in vitro test systems. For example, Morita et al. (1978) reported that juices prepared from some common vegetables, fruits, and spices, including cabbage, broccoli, green pepper, eggplant, apple, shallot, ginger, pineapple, and mint leaf, reduced the mutagenic acti- vity of tryptophan pyrolysates. Lai (1979) and Lai et al. (1980) have also found antimutagenic activity in extracts of wheat sprouts, leaf 13-13

290 DIET, NUTRITION, AND CANCER lettuce, parsley, Brussels sprouts, mustard greens, spinach, cabbage, broccoli, and other vegetables. Although they concluded that the anti- mutagenic substance in these vegetables is chlorophyll, certain food substances without chlorophyll, such as apples, also inhibit mutagenic activity (Morita et al., 1978), indicating that some factor in foods other than chlorophyll can be antimutagenic. Hemin Certain pigments derived from animal systems have also been re- ported to have antimutagenic activity. For example, hemin inhibited the activity of a number of polycyclic mutagens including benzo~a~py- rene, 3-methylcholanthrene, 2-acetylaminofluorene, 2-nitrofluorene, and alfatoxin B1 as well as several mutagenic amino acid pyrolysates, such as Trp-P-1 and Trp-P-2 (Arimoto et al., 1980a,b). The heme metabolites biliverdin and bilirubin also interfered with the mutagenic activity of some of these compounds. The mechanism of these antimutagenic effects awaits complete elucidation. Hemin interfered with the mutagenic activ- ity of 2-nitrofluorene and the activated forms of Trp-P-1 and Glu-P-l, all of which are mutagenic to S. typhimurium in the absence of a mammal- ian metabolic activation system. Therefore, at least some of the anti- mutagenic activity of hemin must be unrelated to such activation. Fatty Acids Other chemicals in foods have also been reported to inhibit muta- genic activity. For example, the unsaturated fatty acids oleic acid and linoleic acid (but not the saturated fatty acids stearic acid and palmitic acid) inhibited the mutagenic activity of a number of chemicals for S. typhimurium (Hayatsu et al., 1981a,b). The mechanism for this inhibition is unknown. Nitrite Yoshida and Matsumoto (1978) reported that when pyrolyzed casein (a mutagenic extract of roasted chicken meat), tobacco-smoke condensate, and certain aromatic amine s were treated with nitrite under acidic con- ditions, there was a decrease in the mutagenic activity of these sub- stances when tested on Salmonella typhimurium. Concentrations of sodium nitrite as low as 3 mg/liter were sufficient to cause a loss of most of the mutagenic activity of casein pyrolysate. Similarly, Tsuda et al. (1980) found that acidic treatment with 2.3 mg/liter solution of sodium nitrite resulted in the loss of mutagenic activity of Trp-P-l, Trp-P-2, and Glu-P-l. A more complex situation has been found to exist for the interaction of nitrite with 2-amino-~-carboline (Tsuda et al., 1981~. At pH of 13-14

Mutagens in Food 291 approximately 4, the reaction results in a loss of mutagenic activity through the conversion of 2-amino-~-carboline to the nonmutagen 2-hydroxy-~-carboline. However, when the pH was below 3.5, a new, direct-acting mutagen was formed: 2-hydroxy-3-nitroso-a-carboline. Thus, nitrite can either neutralize mutagens or result in the forma- tion of new mutagens. The ability of nitrite to interact with dietary amines to form mutagenic and carcinogenic N-nitrosamines is discussed in Chapter 12. Antioxidants A number of antioxidants have been shown to inhibit the mutagenicity of a variety of chemicals. For example, McKee and Tometsko (1979) found that butylated hydroxyanisole (BRA) and butylated hydroxytoluene (BHT) were antimutagenic in the presence of a series of mutagens that require in vitro metabolic activation, but not in the presence of mutagens that are directly mutagenic to S. typhimurium without an added metabolic activation system. Similarly, Katoh et al. (1980) found that BRA in- hibits the mutagenicity of benzo~a~pyrene in Chinese hamster V-79 cells, but not the mutagenicity of the d~rect-acting compound N-acetoxy-2-ace- tylaminofluorene. These findings are consistent with the hypothesis that these antioxidants interfere with the In vitro metabolic activa- tion of the mutagens, rather than reacting with them or their active metabolites directly. Further evidence that a metabolism-modifying mechanism is related to the ability of certain chemicals to exert anticarcinogenic activity is discussed in Chapter 15. The observation that some antioxidant antimutagens appear to act by interfering with metabolic activation does not exclude the possibility that, in some cases, direct reaction with a mutagen may be an important mechanism of action. For example, Shamberger et al. (1979) have found that BHT, ascorbic acid, vitamin E, and selenium can interfere with the mutagenicity of 6-propiolactone and, in some strains of S. typhimurium, malonaldehyde. These two chemicals do not require a mammalian-derived metabolic activation system for activity. Similarly, Guttenplan (1978) attributes the ability of ascorbic acid to inhibit the mutagenic activity of the direct-acting mutagen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) to a direct reaction between ascorbate and the mutagen. Rosin and Stich (1979) reported that some antioxidants, including sodium bisulfite and sodium ascorbate, inhibit the mutagenicity of MING in S. typhimurium, but not that of another direct-acting mutagen, N-acetoxy-2-acetylaminofluorene. Other antioxidant s tested inhibited both or neither of these mutagens. These findings probably reflect an underlying complex mechanism concerning the inhibition by antioxidants of the mutagenicity of even direct-acting mutagens. Retinal (vitamin A alcohol) inhibits the mutagenic activity of 2-aminofluorene and aflatoxin B1, both of which require metabolic 13-15

292 DIET, NUTRITION, AND CANCER activation, but not that of the direct-acting mutagens adriamycin and diepoxybutane (Baird and Birnbaum, 1979; Busk and Ahlborg, 1980~. Similar to the antioxidants discussed above, these results may indicate that retinal inhibits mutagenesis by interfering with metabolic acti- vation rather than by acting as a scavenger of mutagenic chemicals. However, more experimental evidence is needed to clarify this point. SUMMARY AND CONCLUSIONS Considerable attention has recently been directed toward the pre- sence of mutagenic activity in foods. Many vegetables contain muta- genic flavonoids such as quercetin, keempferol, and their glycosides. Furthermore, some substances found in foods can enhance or inhibit the mutagenic activity of other compounds. Mutagens in charred meat and fish are produced during the pyrolysis of proteins that occurs when foods are cooked at very high temperatures. Normal cooking of meat at lower temperatures can also result in the production of mutagens. Smoking of foods as well as charcoal broiling results in the deposition of mutagenic and carcinogenic polynuclear aromatic compounds such as benzo~a~pyrene on the surface of the food. The production of mutations in bacterial or other tests is an indication that a chemical may be carcinogenic in animals. However, many mutagens detected in foods have not been adequately tested for carcinogenicity. Of those that have been tested, the data on the carcinogenicity of the mutagenic flavonol quercetin are conflicting, and several mutagens isolated from pyrolyzed proteins or amino acids appear to be carcinogenic. It is not yet clear to what extent the mutagens produced by pyrolyzing proteins or amino acids are found in normally cooked foods. The finding that some constituents of food can enhance or inhibit the _ vitro mutagenicity of other compounds should not be interpreted as meaning that these compounds would produce the same effects in living animals or humans. If mutagens that are widely distributed in common foods are con- sistently found to cause cancer in animals, many factors should be considered before action is taken to reduce exposure. For example, cooking of meat and fish produces mutagens, but it also destroys pathogenic microorganisms and parasites. Furthermore, some foods con- tain mutagenic flavonoids but also have high nutritional value. 13-1 6

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294 DIET, NUTRITION, AND CANCER Brown, J. P., and P. S. Dietrich. 1979. Mutagenicity of plant flavonols in the Salmonella/mammalian microsome test. Activation of flavonol glycosides by mixed glycosidases from rat cecal bacteria and other sources. Mutat. Res. 66:223-240. Busk, L., and U. G. Ahlborg. 1980. Retinol (vitamin A) as an inhibitor of the mutagenicity of aflatoxin B1. Toxicol. Lett. 6:243-249. Clarke, C. H., and M. J. Wade. 1975. Evidence that caffeine, 8~ethoxy- psoralen and steroidal diamines are frameshift mutagens for E. cold K-12. Mutat. Res. 28:123-125. Commoner, B., A. J. Vithayathil, P. Dolara, S. Nair, P. Madyastha, and G. C. Cuca. 197 8. Formation of mutagens in beef and beef extract during cooking. Science 201: 913-916. Demerec, M., B. Wallace, and E. M. Witkin. 1948. The gene. Carnegie Inst. Washington Yearb. 47:169-176. Demerec, M., G. Bertani, and J. Flint. 1951. A survey of chemicals for mutagenic action on E. colt. Am. Nat. 85:119-136. Dolara, P., B. Commoner, A. Vithayathil, G. Cuca, E. Tuley, P. Madyastha, S. Nair, and D. Kriebel. 1979. The effect of temperature on the formation of mutagens in heated beef stock and cooked ground beef. Mutat. Res. 60: 231-237. Fukuoka, M., K. Yoshihira, S. Natori, K. Sakamoto, S. Iwahara, S. Hosaka, and I. Hirono. 1980. Characterization of mutagenic principles and carcinogenicity test of dill weed and seeds. J. Pharmacobio-Dyn. 3:236-244. Gezelius, K., and N. Fries. 1952. Phage resistant mutants induced in Escherichia cold by caffeine. Hereditas 38:112-114. Glass, E. A., and A. Novick. 1959. Induction of mutation in chlor- amphenicol-inhibited bacteria. J. Bacteriol. 77:10-16. Guttenplan, J. B. 1978. Mechanisms of inhibition by ascorbate of microbial mutagenesis induced by N-nitroso compounds. Cancer Res. 38:2018-2022. Hardigree, A. A., and J. L. Epler. 1978. Comparative mutagenesis of plant flavonoids in microbial systems. Mutat. Res. 58:231-239. Hayatsu, H., S. Arimoto, K. Togawa, and M. Makita. 1981a. Inhibitory effect of the ether extract of human feces on activities of mutagens: Inhibition by oleic and linoleic acids. Mutat. Res. 81:287-293. 13-18

Mutagens in Food 295 Hayatsu, H., K. Inoue, H. Ohta, T. Namba, K. Togawa, T. Hayatsu, M. lIakita, and Y. Wataya. 1981b. Inhibition of the mutagenicity of cooked-beef basic fraction by its acidic fraction. Mutat. Res. 91:437-442. Hirono, I., I. Ueno, S. Hosaka, H. Takanashi, T. Matsushima, T. Sugimura, and S. Natori. 1981. Carcinogenicity examination of quercetin and rutin in ACT rats. Cancer Lett. 13:15-21. Hollstein, M., and J. McCann. 1979. Short-term tests for carcinogens and mutagens. Mutat. Res. 65:133-226. Hosaka, S., T. Matsushima, I. Hirono, and T. Sugimura. 1981. Carcino- genic activity of 3-amino-1 methyl-5H-pyrido[4,3-b~indole (TRP-P-2), a pyrolysis product of tryptophan. Cancer Lett . 13:23-28. Howard, J. W., and T. Fazio. 1980. Analytical methodology and reported findings of polycyclic aromatic hydrocarbons in foods. J. Assoc. Off. Anal. Chem. 63:1077-1104. Inui, N., Y. Nishi, M. M. Hasegawa, and T. Kawachi. 1980. Induction of 8-azaguanine or ouabain resistant somatic mutation of Chinese ham- ster lung cells by treatment with tryptophan pyrolysis products. Cancer Lett. 9:185-189. Ishikawa, T., S. Takayama, T. Kitagawa, T. Kawachi, M. Kinebuchi, N. Matsukura, E. Uchida, and T. Sugimura. 1979. In vivo experiments on tryptophan pyrolysis products. Pp. 159-167 in E. C. Miller, J. A. Miller, I. Hirono, T. Sugimura, and S. Takayama, eds. Naturally Occurring Carcinogens-Mutagens and Modulators of Carcinogenesis. Japan Scientific Societies Press, Tokyo; University Park Press, Baltimore, Md. Johnson, H. G., and M. K. Bach. 1965. Apparent suppression of mutation rates in bacteria by spermine. Nature 208:408-409. Kasai, H., S. Nishimura, M. Nagao, Y. Takahashi, and T. Sugimura. 1979. Fractionation of a mutagenic principle from broiled fish by high- pressure liquid chromatography. Cancer Lett. 7:343-348. Kasai, H., Z. Yamaizumi, K. Wakabayashi, M. Nagao, T. Sugimura, S. Yokoyama, T. Miyazawa, N. E. Spingarn, J. H. Weisburger, and S. Nishimura. 1980a. Potent novel mutagens produced by broiling fish under normal conditions. Proc. Jpn. Acad. 56(B):278-283. Kasai, H., Z. Yamaizumi, K. Wakabayashi, M. Nagao, T. Sugimura, S. Yokoyama, T. Miyazawa, and S. Nishimura. 1980b. Structure and chemical synthesis of Me-IQ, a potent mutagen isolated from broiled fish. Chem. Lett. 11:1391-1394. 13-19

296 DIET, NUTRITION, AND CANCER Kasai, H., S. Nishimura, K. Wakabayashi, M. Nagao, and T. Sugimura. 1980c. Chemical synthesis of 2-amino-3-methylimidazo[4,5-fJquino- line (IQj, a potent mutagen isolated from broiled fish. Proc. Jpn. Acad. 56(B):382-384. Kasai, H., Z. Yamaizumi, T. Shiomi, S. Yokoyama, T. Miyazawa, K. Wakabayashi, M. Nagao, T. Sugimura, and S. Nishimura. 1981. Struc- ture of a potent mutagen isolated from fried beef. Chem. Lett. 4: 485-488. Katoh, Y., M. Tanaka, K. Umezawa, and S. Takayama. 1980. Inhibition of mutagenesis in Chinese hamster V-79 cells by antioxidants. Toxicol. Lett. 7:125-130. Koehler, P. E., M. E. Mason, and J. A. Newell. 1969. Formation of pyra- zine compounds in sugar-amino acid model systems. J. Agric. Food Chem. 17:393-396. Krone, C. A., and W. T. Iwaoka. 1981. Mutagen formation during the cooking of fish. Cancer Lett. 14:93-99. Kubitschek, H. E., and H. E. Bendigkeit. 1958. Delay in the appearance of caffeine-induced T5 resistance in Escherichia coli. Genetics 43:647-661. Kubitschek, H. E., and H. E. Bendigkeit. 1964. Mutation in continuous cultures. I. Dependence of mutational response upon growth-limiting factors. Mutat. Res. 1:113-120. Lai, C.-N. 1979. Chlorophyll: The active factor in wheat sprout extract inhibiting the metabolic activation of carcinogens ~n vitro. Nutr. Cancer 1~3~:19-21. Lai, C.-N., M. A. Butler, and T. S. Matney. 1980. Antimutagenic activi- ties of common vegetables and their chlorophyll content. Mutat. Res. 7 7: 245-250. Lee, J. S. K., and L. Y. Y. Fong. 1979. Mutagenicity of Chinese alco- holic spirits. Food Cosmet. Toxicol. 17 :575-578. Lijinsky, W., and A. E. Ross. 1967. Production of carcinogenic polynu- clear hydrocarbons in the cooking of food. Food Cosmet. Toxicol. 5: 343-347. Li jinsky, W.t, and P. Shubik. 1964. Benzo~a~pyrene and other poly- nuclear hydrocarbons in charcoal-broiled meat. Science 145: 53-55. Lintas, C., M. C. De Matthaeis, and F. Merli. 1979. Determination of benzo~a~pyrene in smoked, cooked and toasted food products. Food Cosmet. Toxicol. 17: 325-328. 13-20

Mutagens in Food 297 Loquet, C., G. Toussaint, and J. Y. LeTalaer. 1981. Studies on muta- genic constituents of apple brandy and various alcoholic beverages collected in Western France, a high incidence area for oesophageal cancer. Mutat. Res. 88:155-164. MacGregor, J. T., and L. Jurd. 1978. Mutagenicity of plant flavo- noids: Structural requirements for mutagenic activity in Salmo- nella typhimurium. Mutat. Res. 54:297-309. Maga, J. A. 1979. Furans in foods. CRC Crit. Rev. Food Sci. Nutr. 11:355-400. Maruta, A., K. Enaka, and M. Umeda. 1979. Mutagenicity of quercetin and kaempferol on cultured mammalian cells. Gann 70:273-276. Masuda, Y., K. Mori, and M. Kuratsune. 1966. Polycyclic aromatic hydrocarbons in common Japanese foods. I. Broiled fish, roasted barley, shoyu, and caramel. Gann 57:133-142. Matsukura, N., T. Kawachi, K. Morino, H. Ohgaki, T. Sugimura, and S. Takayama. 1981a. Carcinogenicity in mice of mutagenic com- pounds from a tryptophan pyrolyzate. Science 213:346-347. Matsukura, N., T. Kawachi, K. Wakabayashi, H. Ohgaki, K. Morino, T. Sugimura, H. Nukaya, and T. Kosuge. 1981b. Liver cancer and pre- cancerous changes in rats induced by the basic fraction of tryptophan pyrolysate. Cancer Lett. 13:181-186. Matsumoto, T., D. Yoshida, S. Mizusaki, and H. Okamoto. 1977. Muta- genic activity of amino acid pyrolyzates in Salmonella typhimurium TA 98. Mutat. Res. 48:279-286. Matsumoto, T., D. Yoshida, S. Mizusaki, and H. Okamoto. 1978. Muta- genicities of the pyrolyzates of peptides and proteins. Mutat. Res. 56:281-288. Matsumoto, T., D. Yoshida, and H. Tomita. 1981. Determination of mutagens, amino~x-carbolines in grilled foods and cigarette smoke condensate. Cancer Lett. 12:105-110. McCann, J., and B. N. Ames. 1976. Detection of carcinogens as muta- gens in the Salmonella/microsome test: Assay of 300 chemicals: Discussion. Proc. Natl. Acad. Sci. U.S.A. 73:950-954. McCann, J., E. Choi, E. Yamasaki, and B. N. Ames. 1975. Detection of carcinogens as mutagens in the Salmonella/microsome test: Assay of 300 chemicals. Proc. Natl. Acad. Sci. U.S.A. 72:5135-5139. 13-21

298 DIET, NUTRITION, AND CANCER McKee, R. H., and A. M. Tometsko. 1979. Inhibition of promutagen acti- vation by the antioxidants butylated hydroxyanisole and butylated hydroxytoluene. J. Natl. Cancer Inst. 63: 473-47 7. Meltz, M. L., and J. T. MacGregor. 1981. Activity of the plant flavanol quercetin in the mouse lymphoma L5178Y TO /- mutation, DNA single- strand break, and Balb/c 3T3 chemical transformation assays. Mutat. Res. 88:317-324. Morino, K., N. Matsukura, T. Kawachi, H. Ohgaki, T. Sugimura, and I. Hirono. 1982. Carcinogenicity test of quercetin and rutin in golden hamsters by oral administration. Carcinogenesis 3:93-97. Morita, K., M. Hara, and T. Kada. 1978. Studies on natural desmutagens: Screening for vegetable and fruit factors active in inactivation of mutagenic pyrolysis products from amino acids. Agric. Biol. Chem. 42:1235-1238. National Cancer Institute. 1978. Bioassay of Aniline Hydrochloride for Carcinogenicity. Carcinogenesis Technical Report Series No. 130. DHEW Publication No. (NIH)78-1385. Carcinogenesis Testing Program, National Cancer Institute, Bethesda, Md. 109 pp. Nagao, M., M. Honda, Y. Seino, T. Yahagi, and T. Sugimura. 1977a. Muta- genicities of smoke condensates and the charred surface of fish and meat. Cancer Lett. 2:221-226. Nagao, M., M. Honda, Y. Seino, T. Yahagi, T. Kawachi, and T. Sugimura. 1977b. Mutagenicities of protein pyrolysates. Cancer Lett. 2: 335-340. Nagao, M., T. Yahagi, T. Kawachi, Y. Seino, M. Honda, N. Matsukura, T. Sugimura, K. Wakabayashi, K. Tsuji, and T. Kosuge. 1977c. Mutagens in foods, and especially pyrolysis products of protein. Pp. 259-264 in D. Scott, B. A. Bridges, and F. H. Sobels, eds. Progress in Genetic Toxicology. Elsevier/North-Holland, New York, Amsterdam, and Oxford. Nagao, M., T. Yahagi, T. Sugimura, T. Kosuge, K. Tsuji, K. Wakabayashi, S. Mizusakai, and T. Matsumoto. 1977d. Comutagenic action of norharman and harman. Proc. Jpn. Acad. 53~2~:95-98. Nagao, M., T. Yahagi, M. Honda, Y. Seino, T. Matsushima, and T. Sugimura. 1977e. Demonstration of mutagenicity of aniline and o-toluidine by norha~an. Proc. Jpn. Acad. 53(B)~:34-37. Nagao, M., Y. Takahashi, H. Yamanaka, and T. Sugimura. 1979. Mutagens in coffee and tea. Mutat. Res. 68:101-106. 13-22

Mutagens in Food 299 Nagao, M., Y. Takahashi, K. Wakabayashi, and T. Sugimura. 1981. Mutagenicity of alcoholic beverages. Mutat. Res. 88:147-154. Novick, A. 1956. Mutagens and antimutagens. Brookhaven Symp. Biol. 8:201-215. Pamuckcu, A. M., §. Yal~iner, J. F. Hatcher, and G. T. Bryan. 1980. Quercetin, a rat intestinal and bladder carcinogen present in bracken fern (Pteridium aquilinum). Cancer Res. 40:3468-3472. Poindexter, E. H., Jr., and R. D. Carpenter. 1962. The isolation of harmane and norharmane from tobacco and cigaret smoke. Phytochem- istry 1:215-221. Powrie, W. D., C. H. Wu, M. P. Rosin, and H. F. Stich. 1981. Clasto- genic and mutagenic activities of Maillard reaction model systems. J. Food Sci. 46:1433-1438, 1445. Purchase, I. F. H., E. Longstaff, J. Ashby, J. A. Styles, D. Anderson, P. A. Lefevre, and F. R. Westwood. 1978. An evaluation of 6 short- term tests for detecting organic chemical carcinogens. Br. J. Cancer 37:873-959. Rosin, M. P., and H. F. Stich. 1979. Assessment of the use of the Sal- monella mutagenesis assay to determine the influence of antioxidants on carcinogen-induced mutagenesis. Int. J. Cancer 23:722-727. Saito, D., A. Shirai, T. Matsushima, and I. Hirono. 1980. Test of car- cinogenicity of quercetin, a widely distributed mutagen in food. Teratog., Carcinog., Mutagen. 1:213-221. Seino, Y., M. Nagao, T. Yahagi, T. Sugimura, T. Yasuda, and S. Nishimura. 1978. Identification of a mutagenic substance in a spice, sumac, as quercetin. Mutat. Res. 58:225-229. Seppilli, A., and G. S. Sforzolini. 1963. [In Italian.] Sulla presenza di idrocarburi policiclici cancerigeni nelle carni cotte alla graticola. Boll. Soc. Ital. Biol. Sper. 39:110-111. Shamberger, R. J., C. L. Corlett, K. D. Beaman, and B. L. Kasten. 1979. Antioxidants reduce the mutagenic effect of malonaldehyde and S-pro- piolactone. Part IX, Antioxidants and cancer. Mutat. Res. 66:349-355. Shinohara, K., R.-T. Wu, N. Jahan, M. Tanaka, N. Morinaga, H. Murakami, and H. Omura. 1980. Mutagenicity of the browning mixtures by amino- carbonyl reactions on Salmonella typhimurium TA 100. Agric. Biol. Chem. 44:671-672. 13-23

300 DIET, NUTRITION, AND CANCER Simmon, V. F. 1979. In vitro mutagenicity assays of chemical carcino- gens and related compounds with Salmonella typhimurium. J. Natl. Cancer Inst. 62:893-899. Spingarn, N. E., and C. T. Garvie. 1979. Formation of mutagens in sugar-ammonia model systems. J. Agric. Food Chem. 27:1319-1321. Spingarn, N., E., L. A. Slocum, and J. H. Weisburger. 1980a. Formation of mutagens in cooked foods. II. Foods with high starch content. Cancer Lett. 9:7-12. Spingarn, N. E., H. Kasai, L. L. Vuolo, S. Nishimura, Z. Yamaizumi, T. Sugimura, T. Matsushima, and J. H. Weisburger. 1980b. Formation of mutagens in cooked foods. III. Isolation of a potent mutagen from beef. Cancer Lett. 9:177-183. Stich, H. F., W. Stich, M. P. Rosin, and W. D. Powrie. 1980. Mutagenic activity of pyrazine derivatives: A comparative study with Salmonella typhimurium, Saccharomyces cerevisiae, and Chinese hamster ovary cells. Food Cosmet. Toxicol. 18:581-584. Stich, H. F., M. P. Rosin, C. H. Wu, and W. D. Powrie. 1981a. Clasto- genicity of furans found in food. Cancer Lett. 13:89-95. Stich, H. F., W. Stich, M. P. Rosin, and W. D Powrie. 1981b. Clasto- genic activity of caramel and caramelized sugars. Mutat. Res. 91:129-136. Stoltz, D. R., B. Stavric, R. Klassen, and T. Muise. In press a. The health significance of mutagens in foods. In H. F. Stich, ed. Carcin- ogens and Mutagens in the Environment. Volume 1, Food Products. CRC Press, Boca Raton, Fla. Stoltz, D. R., B. Stavric, D. Krewski, R. Klassen, R. Bendall, and B. Junkins. In press b. Mutagenicity screening of foods. I. Results with beverages. Environ. Mutagen. Sugimura, T. In press. Food-born genotoxins. In R. Fleck and A. Hollaender, eds. Genetic Toxicology: An Agricultural Perspective. Plenum Press, New York. Sugimura, T., and M. Nagao. In press. The use of mutagenicity to evaluate carcinogenic hazards in our daily lives. In J. A. Haddle, ed. Muta- genicity: New Horizons in Genetic Toxicology. Academic Press, New York. Sugimura, T., S. Sato, M. Nagao, T. Yahagi, T. Matsushima, Y. Seino, M. Takeuchi, and T. Kawachi. 1976. Overlapping of carcinogens and mutagens. Pp. 191-213 in P. N. Magee, S. Takayama, T. Sugimura, and T. Matsuhima, eds. Fundamentals in Cancer Prevention. University Park Press, Baltimore, London, and Tokyo. 13-24

Mutagens in Food 301 Sugimura, T., T. Kawachi, M. Nagao, T. Yahagi, Y. Seino, T. Okamoto, K. Shudo, T. Kosuge, K. Tsuji, K. Wakabayashi, Y. Iitaka, and A. Itai. 1977. Mutagenic principled) in tryptophan and phenylalanine pyrolysis products. Proc. Jpn. Acad. 53~1) :58-61. Sugimura, T., M. Nagao, and K. Wakabayashi. 1982. The metabolic aspects of the comutagenic action of norharman. Pp. 1011-1025 in R. Synder, D. Parke, J. J. Kocsis, D. J. Jollow, C. G. Gibson, and C. M. Witmer, eds. Biological Reactive Intermediates 2. Part B. Plenum Press, New York. Takahashi, Y., M. Nagao, T. Fujino, Z. Yamaizumi, and T. Sugimura. 1979. Mutagens in Japanese pickle identified as flavonoids. Mutat. Res. 68:117-123. Takase, S., and H. Murakami. 1966. Fluorescence of sake. I. Fluores- cence spectrum of sake and identification of barman. Agric. Biol. Chem. 30:869-876. Takayama, S., Y. Katoh, M. Tanaka, M. Nagao, K. Wakabayashi, and T. Sugimura. 1977. In vitro transformation of hamster embryo cells with tryptophan pyrolysis products. Proc. Jpn. Acad. 53(B):126-129. Takayama, S., T. Hirakawa, and T. Sugimura. 1978. Malignant trans- formation in vitro by tryptophan pyrolysis products. Proc. Jpn. Acad. 54(B):418 - 22. Takayama, S., T. Hirakawa, M. Tanaka, T. Kawachi, and T. Sugimura. 1979. _ vitro transformation of hamster embryo cells with a glutamic acid pyrolysis product. Toxicol. Lett. 4 :281-284. Takeda, K., T. Ohta, K. Shudo, T. Okamoto, K. Tsuji, and T. Kosuge. 1977. Synthesis of a mutagenic principle isolated from tryptophan pyrolyzate. Chem. Pharm. Bull. 25:2145-2146. Takeuchi, T., K. Ogawa, H. Iinuma, H. Suda, K. Ukita, T. Nagatsu, M. Kato, and H. Umezawa. 1973. Monoamine oxidase inhibitors isolated from fermented broths. J. Antibiot. 26:162-167. Tamura, G., C. Gold, A. Ferro-Luzzi, and B. N. Ames. 1980. Fecalase: A model for activation of dietary glycosides to mutagens by intes- tinal flora. Proc. Natl. Acad. Sci. U.S.A. 77:4961-4965. Tohda, H., A. Oikawa, T. Kawachi, and T. Sugimura. 1980. Induction of sister-chromatic exchanges by mutagens from amino acid and protein pyrolysates. Mutat. Res. 77:65-69. Tsuda, M., Y. Takahashi, M. Nagao, T. Hirayama, and T. Sugimura. 1980. Inactivation of mutagens from pyrolysates of tryptophan and glutamic acid by nitrite in acidic solution. Mutat. Res. 78:331-339. 13-25

302 DIET, NUTRITION, AND CANCER Tsuda, M., M. Nagao, T. Hirayama, and T. Sugimura. 1981. Nitrite con- converts 2-amino-a-carboline, an indirect mutagen, into 2-hydroxy-~- carboline, a non-mutagen, and 2-hydroxy-3-nitroso-~-carboline, a direct mutagen. Mutat. Res. 83:61-68. Umezawa, K., A. Shirai, T. Matsushima, and T. Sugimura. 1978. Comu- tagenic effect of norharman and barman with 2-acetylaminofluorene derivatives. Proc. Natl. Acad. Sci. U.S.A. 75:928-930. Uyeta, M., T. Kanada, M. Mazaki, S. Taue, and S. Takahashi. 1979. Assaying mutagenicity of food pyrolysis products using the Ames test. Pp. 169-176 in E. C. Miller, J. A. Miller, I. Hirono, T. Sugimura, and S. Takayama, eds. Naturally Occurring Carcinogens- Mutagens and Modulators of Carcinogenesis. Japan Scientific Societies Press, Tokyo; University Park Press, Baltimore, Md. Uyeta, M., S. Taue, and M. Mazaki. 1981. Mutagenicity of hydrolysates of tea infusions. Mutat. Res. 88:233-240. Vithayathil, A. J., B. Commoner, S. Nair, and P. Madyastha. 1978. Isola- tion of mutagens from bacterial nutrients containing beef extract. J. Toxicol. Environ. Health 4:189-202. Wakabayashi, K., K. Tsuji, T. Kosuge, K. Takeda, K. Yamaguchi, K. Shudo, Y. Iitaka, T. Okamoto, T. Yahagi, M. Nagao, and T. Sugimura. 1978. Isolation and structure determination of a mutagenic substance in L-lysine pyrolysate. Proc. Jpn. Acad. 54(B):S69-571. Wakabayashi, K., M. Nagao, T. Kawachi, and T. Sugimura. 1981. Co-muta- genic effect of norharman with N-nitrosamine derivatives. Mutat. Res. 80:1-7. Watson, W. A. F. 1982. The mutagenic activity of quercetin and kaempferol in Drosophila melanogaster. Mutat. Res. 103:145-147. Weisburger, J. H., and N. E. Spingarn. 1979. Mutagens as a function of mode of cooking of meats. Pp. 177-184 in E. C. Miller, J. A. Miller, I. Hirono, T. Sugimura, and S. Takayama, eds. Naturally Occurring Carcinogens-Mutagens and Modulators of Carcinogenesis. Japan Scien- tific Societies Press, Tokyo; University Park Press, Baltimore, Md. Yamaguchi, K., H. Zenda, K. Shudo, T. Kosuge, T. Okamoto, and T. Sugimura. 1979. Presence of 2-aminodipyrido[1,2-a:3',2'-diimidazole in casein pyrolysate. Gann 70:849-850. Yamaguchi, K., K. Shudo, T. Okamoto, T. Sugimura, and T. Kosuge. 198Oa. Presence of 3-amino-1,4-dimethyl-5H-pyrido[4,3-b~indole in broiled beef. Gann 71:745-746. 13-26

Mutagens in Food 303 Yamaguchi, K., K. Shudo, T. Okamoto, T. Sugimura, and T. Kosuge. 1980b. Presence of 2-aminodipyrido[1,2-a:3'2'-d~imidazole in broiled cuttle- fish. Gann 71:743-744. Yamaizumi, Z., T. Shiomi, H. Kasai, S. Nishimura, Y. Takahashi, M. Nagao, and T. Sugimura. 1980. Detection of potent mutagens, Trp-P-1 and Trp-P-2, in broiled fish. Cancer Lett. 9:75-83. Yamamoto, T., K. Sutji, T. Kosuge, T. Okamoto, K. Shudo, K. Takeda, Y. Iitaka, K. Yamaguchi, Y. Seino, T. Yahagi, M. Nagao, and T. Sugimura. 1978. Isolation and structure determination of mutagenic substances in L-glutamic acid pyrolysate. Proc. Jpn. Acad. 54(B):248-250. Yasuda, T., Z. Yamaizumi, S. Nishimura, M. Nagao, Y. Takahashi, H. Fujiki, T. Sugimura, and K. Tsuji. 1978. Detection of comutagenic compounds, harman and norharman in pyrolysis product of proteins and food by gas chromatograph-mass spectrometry. Nippon Gan Gakkai Sokai Kiji 37:6~. Abstract 41. Yoshida, D., and T. Matsumoto. 1978. Changes in mutagenicity of protein pyrolyzates by reaction with nitrite. Mutat. Res. 58:35-40. Yoshida, D., and H. Okamoto. 1980. Formation of mutagens by heating the aqueous solution of amino acids and some nitrogenous compounds with addition of glucose. Agric. Biol. Chem. 44:2521-2522. Yoshida, D., T. Matsumoto, R. Yoshimura, and T. Matsuzaki. 1978. Muta- genicity of amino-a-carbolines in pyrolysis products of soybean globulin. Biochem. Biophys. Res. Commun. 83:915-920. 13-27

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Based on a thorough review of the scientific evidence, this book provides the most authoritative assessment yet of the relationship between dietary and nutritional factors and the incidence of cancer. It provides interim dietary guidelines that are likely to reduce the risk of cancer as well as ensure good nutrition.

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