Food-Derived Mutagens and Carcinogens1

[CANCER RESEARCH (SL'PPL.) 52. 2092s-2098s. April I, 1992] Food-derived Mutagens and Carcinogens1

Keiji Wakabayashi,2 Minako Nagao, Hiroyasu Esumi, and Takashi Sugimura

National Cancer Center Research Institute, l-l, Tsukiji 5-chome, Chuo-ku, Tokyo 104, Japan

Abstract pounds have planar structures that can be inserted between the base pairs of double-stranded DNA. Charred parts of broiled Cooked food contains a variety of mutagenic heterocyclic amines. All the mutagenic heterocyclic amines tested were carcinogenic in rodents fish and meat were also found to be mutagenic to TA98 after when given in the diet at 0.01-0.08%. Most of them induced cancer in metabolic activation (9, 10). These findings led to the isolations the liver and in other organs. It is noteworthy that the most abundant of the mutagenic compounds, IQ,' MelQ, MelQx from broiled heterocyclic amine in cooked food, 2-amino-l-methyl-6-phenylimi- fish and meat (5-7). Later PhIP was also isolated as member dazo(4,5-A|pyridine, produced colon and mammary carcinomas of this class of mutagens (11). These heterocyclic amines were in rats and lymphomas in mice but no hepatomas in either. 2-Amino-3- methylimidazo(4,5-/]quinoline induced liver cancer in monkeys. Forma formed by heating mixtures of creatinine, sugars, and amino acids, which are present in raw meat and fish (12-16). More tion of adducts with guanine by heterocyclic amines is presumably involved in their carcinogenesis. Quantification of heterocyclic amines in recently identified mutagens have been found to contain oxygen cooked foods and in human urine indicated that humans are continuously atoms (17-19). Before the discoveries of imidazoquinoline and exposed to low levels of them in the diet. These low levels of heterocyclic imidazoquinoxaline compounds, pyrolysates of food compo amines are probably insufficient to produce human cancers by themselves. nents, amino acids, and proteins were shown to contain muta However, a linear relationship between DNA adduct levels and a wide genic substances. These findings led to the identification of range of doses of a heterocyclic amine was demonstrated in animals. It pyridoindole, dipyridoimidazole, phenylpyridine, and tetraaza- suggests that even very low doses of heterocyclic amines form DNA adducts and may be implicated in the development of human cancer under fluoranthene derivatives as new mutagenic heterocyclic amines conditions in which many other mutagens-carcinogens, tumor promoters, in pyrolysates of amino acids and of amino-a-carboline deriva and factors stimulating cancer progression exist. tives as mutagenic compounds in a pyrolysate of protein (4-7). Some of the heterocyclic amines that were originally isolated Introduction from pyrolysates of amino acids and proteins were also re covered from cooked foods (6). Diet plays an important role in cancer development: high intakes of total calories and fat enhance cancer development, excessive intake of sodium chloride promotes carcinogenesis in Chemistryof Heterocyclic Amines in Foods the stomach, whereas, in contrast, fiber, carotene, and vitamins protect against cancer development (1, 2). Foods also contain The heterocyclic amines reported so far are listed in Table 1 mutagenic and/or carcinogenic substances as very minor com with their chemical names, abbreviations, and sources (11, 17, ponents: mycotoxin contaminants, nitrosamines, polycyclic ar 18, 20-29). They are classified as IQ-type compounds, in which omatic hydrocarbons, and plant alkaloids (3). the amino group is not changed by treatment with 2 HIMsodium A series of mutagenic-carcinogenic substances have been nitrite, and as non-IQ-type compounds, in which the amino found in cooked meat and fish in the last 15 years. New group is converted to a hydroxy group with 2 HIMsodium nitrite heterocyclic amine compounds were isolated by monitoring the (30). The amino group of IQ-type heterocyclic amines is con fractionation and purification of mutagens by using microbial verted to a nitro group with 50 mM sodium nitrite and the mutation assays (4-7). These compounds have much higher mutagenic activity than other typical mutagens-carcinogens and resulting compound shows a similar mutagenicity to the origi nal compound in the absence of S9 mix (31). Most of these were later shown to be carcinogenic in rodents and nonhuman heterocyclic amines have been synthesized in large quantities primates. This review deals mainly with mutagenic and carcin for use in long-term experiments in rodents (6, 32). These ogenic heterocyclic amines that are produced under ordinary cooking conditions. compounds remain stable in experimental diets if these diets In 1939, Widmark (8) reported that organic solvent extracts are kept cold and dry. They are also sufficiently stable in dilute of broiled horse meat induced tumors in mammary glands when aqueous solution for biological experiments. repeatedly painted on the backs of mice. In the 1970s we became The structures of heterocyclic amines are shown in Fig. 1. A interested in whether the smoke produced by broiling meat and heterocyclic amine containing oxygen, namely aminomethylim- fish contained mutagenic and carcinogenic activity, because idazofuropyridine, can exist as four possible isomers because cigarette smoke condensate was known to contain many muta there are two possible positions for both the oxygen and N- gens-carcinogens. We found that a smoke condensate obtained methyl group (17). A mutagenic heterocyclic amine containing by broiling fish and trapping the smoke on glass fiber filters oxygen atoms was also isolated from a creatine pyrolysate and exerted mutagenic activity toward Salmonella typhimurium identified as Cre-P-1 (Fig. 1) (18). However, the presence of TA98 after microsomal metabolic activation (9, 10). TA98 was Cre-P-1 in cooked food has not yet been reported. more sensitive than TA 100, indicating that the mutagenic com- 3The abbreviations used are: IQ. 2-amino-3-methylimidazo[4,5-/)quinoline; 1Presented at "Nutrition and Cancer," the first conference of the International MelQ. 2-amino-3.4-dimethylimidazo[4,5-/]quinoline; MelQx, 2-amino-3,8-di- Conference Series on Nutrition and Health Promotion. April 17-19. 1991. methylimidazo|4,5-/|quinoxaline; PhIP, 2-amino-1 -methyl-o-phenylimidazo^.S- Atlanta, GA. This study was supported by Grams-in-Aid for Cancer Research Ajpyridine; AnC. 2-amino-9//-pyrido[2,3-ft]indole; MeAaC, 2-amino-3-methyl- from the Ministry of Health and Welfare and the Ministry of Education. Science 9//-pyrido[2,3-6]indole; Trp-P-2.3-amino-1 -methyl-5//-pyrido|4,5-e]indole; Glu- and Culture of Japan, and a Grant-in-Aid from the Ministry of Health and P-1. 2-amino-6-methyldipyrido(1,2-a:3',2'-

Table 1 Chemical names, abbreviations, and sources of isolated helerocyclic amines and references name2-Amino-3-methylimidazo[4.5-/]quinoline2-Amino-3,4-dimethylimidazo|4.5-/)quinoline2-Amino-3-methylimidazo|4,5-./]quinoxaline2-Amino-3.8-dimethylimidazo[4.5-/]quinoxaline2-Amino-3,4,8-trimelhylimidazo[4,5,-./]quinoxaline2-Amino-3.7,8-trimethylimidazo[4,5-/)quinoxaline2-Amino-Chemical sardineBroiledsun-dried sardineCreatine-supplememedsun-dried friedNorwegian productFried meat beefHeated creatinine.threonine.mixture of glucoseHeatedand creatinine.glycine,mixture of glucoseFriedand -methyl-o-phenylimidazo^.S-Alpyridine2-Amino-n.n.n-trimethylimidazopyridine2-Amino-n.n-dimelhylimidazopyridine2-Amino-(l1 beefCreatine-supplcmented friedNorwegian productCreatine-supplementedmeat friedNorwegian productFried meat 3,2)-e|imi-dazo[4.5-ft]p>or 3).6-dimelhylfuro[2.3(or mixture of beef, crea ridine4-Amino- milkCreatinetine, and W.6W-pyr-rolo[3.4-/)benzimidazole-5.7-dione3-Amino-1,6-dimethyl-2-methylamino- 1 pyrolysateTryptophan

,4-dimethyl-5//-pyrido[4.3-6]indole3-Amino-1 pyrolysateTryptophan -methyl-5//-pyrido(4,3-o]indole2-Amino-6-methyldipyrido[l,2-a:3'.2'-

Mutagenesis gland cancers in female rats (47), and lymphomas in mice of both sexes (48). As described briefly in the introduction, heterocyclic amines Injection s.c. of Trp-P-1 resulted in the formation of fibro- are more mutagenic toward 5. typhimurium TA98 than TA 100. sarcomas at the injection site in F344 rats and Syrian golden The specific mutagenicities of some heterocyclic amines are hamsters (54). Intragastric intubation of IQ resulted in devel much stronger than those of typical mutagens-carcinogens such opment of tumors in the mammary gland, liver, and ear duct as aflatoxin B,, 4-nitroquinoline 1-oxide, and benzo[a]pyrene. of SD rats (55). Intestinal tumors developed much more rapidly Moreover, imidazoquinoline and imidazoquinoxaline deriva in analbuminemic congenie F344 strain rats (F344-Ã›/A rats) tives, including IQ, MelQ, and MelQx are much more muta than in F344 rats, when IQ was given with the diet.4 Albumin- genic than AaC, MeAaC, and PhIP, as shown in Table 2 (4- deficient SD rats (NAR rats) were also very sensitive to induc 7). However, these marked differences between mutagenicities tion of intestinal tumors by PhIP (56). Hepatocellular carci of heterocyclic amines and other typical mutagens-carcinogens nomas also developed in cynomolgus monkeys (Macaca fasci- were not found in cultured mammalian cell systems, such as cularis) after repeated administration of IQ by gavage (57). Chinese hamster lung cells with diphtheria toxin resistance (33) and repair-deficient Chinese hamster ovary cells with 6-thiog- uanine resistance (34, 35). Results with these mammalian mu Metabolism of Heterocyclic Amines and Their Modification tation systems closely reflect carcinogenic potencies, the carcin of DNA ogenic potencies of IQ, MelQ, and MelQx being almost the Heterocyclic amines are metabolically activated by cyto- same as those of AaC, MeAaC, and PhIP. One factor respon chrome P450s with conversion of an amino group to a hydrox- sible for the difference between bacterial systems and mamma yamino group (58-63). The principal cytochrome P450 isozyme lian systems would be esterifying enzymes such as acetyltrans- responsible for this conversion is induced in rat liver by 3- ferase and sulfotransferase. Heterocyclic amines induce chro methylcholanthrene. Recent investigations using cloned com mosomal aberrations (36) and sister chromatid exchanges (34, 37-39) in cultured cells in vitro. Heterocyclic amines were also plementary DNAs for various molecular species of cytochrome P450s driven by vaccinia virus expression vector indicated that positive in a wing spot test with Drosophila melanogaster (40). cytochrome P450IA2 is the most effective for metabolic acti vation of heterocyclic amines (64). Hydroxyamino derivatives Carcinogenesis of heterocyclic amines are further activated by forming esters Long-term animal experiments with rats and mice fed a diet with acetic acid, sulfuric acid, and proline. These esters may be containing heterocyclic amines have been carried out, in most the ultimate forms producing DNA adducts (58, 61, 65). The cases with the maximum tolerable doses (MTD) of the com formation of the IQ-guanine adduci is shown schematically in pounds. The results are summarized in Table 3 (41-53). In Fig. 2 (66). The structures of guanine adducts with Trp-P-2 and BALB/c x DBA/2 F, mice tumors developed in the liver, Glu-P-1 were also elucidated (67, 68). These DNA adducts forestomach, lung, blood vessels, hematopoietic system, and should be responsible for the genetic alteration leading to lymphoid tissue (42,44,46, 48, 50, 52, 53). In F344 rats tumors carcinogenesis. In vitro experiments using the acetoxyamino developed in the liver, intestine, skin, oral cavity, mammary derivative of Glu-P-1 and a plasmid containing the human c- gland, Zymbal gland, and clitoral gland (41, 43, 45, 47, 49, 51, Ha-ras-1 protooncogene sequence demonstrated the hot spots 53). Most heterocyclic amines were hepatocarcinogens. How for mutation in the region of the CCGG sequence, correspond ever, PhIP did not induce hepatomas in rats or mice but instead ing to codons il (GCC) and 12 (GGC) (69). The c-Ha-ras induced colon cancers preferentially in male rats, mammary 4M. Ochiai el al., manuscript in preparation. 2093s

adducts as those in the liver, but these organs show no histo lÃ³gica!precancerous changes, although nephro- and cardiolox- icological effects might appear much later (74, 75). Further

IQ MelQ IQx more, the formation of DNA adducts alone may not be suffi cient to produce cancers. This is suggested from a linear relation NH, NH, N=< N=< found between levels of MelQx-DNA adducts in the liver of 1 N-CH, ' N-CH, rats and doses ranging from 0.4 ppm to the dose used in a carcinogenicity test, 400 ppm (75). However, the hepalocarcin- MelQx 4.8-DiMelQx 7.8-DiMelQx ogenic response of mice to various doses (20, 60, 200, and 600 ppm) of MelQx seemed to be nonlinear (76). These results suggest that the genetic alterations produced by heterocyclic amines are enhanced by liver regeneration induced by hetero cyclic amines themselves at higher doses. The linearity of the PhIP TMIP DMIP dose response for carcinogenesis in the intestine and other organs has not been examined. The pharmacokinetics of heterocyclic amines are important. Ingested heterocyclic amines are absorbed from the intestine, and their metabolic activation and inactivation occur mainly in the liver. Hydroxylation of ring carbons followed by conjugation MelFP Cre-P-1 leads to their inactivation, and detoxified forms of heterocyclic amines are excreted in the urine and feces (77-79). However, the pattern of in vivo metabolism differs for different hetero cyclic amines. PhIP is absorbed from the intestine and most of CH, it is excreted as PhIP in the bile and feces in rats (80). However, Trp-P-1 Trp-P-2 Glu-P-1 in mice treated with polychlorinated biphenyls, little unmeta- bolized PhIP is detected in the feces (81).

Quantification of Heterocyclic Amines and Consideration of Glu-P-2 Phe-P-1 Their Risk for Cancer Development Orn-P-1 Methods have been developed for quantitating heterocyclic amines in various materials such as cooked foods, cigarette smoke condensate, and urine (6,82-84). The procedures consist of blue-cotton treatment, HCl-methylene dichloride partition, AoC MeAÃŸC and conventional and high-performance liquid chromatogra- Fig. I. Structures of mutagenic and carcinogenic heterocyclic amines. See phy. An electrochemical method is effective for detecting imi- Table 1 for abbreviations. dazoquinoline and imidazoquinoxaline derivatives (82). Some examples of analytical data are given in Table 4 (6, 76). PhIP mutations were frequently observed in squamous cell carcino Table 2 Mutagenicilies of heterocyclic amines and typical carcinogens in mas of the Zymbal gland of rats induced by IQ, MelQ, and Salmonella typhimurium TA98 and TAIOO" MelQx (70). Most of the mutations were found in codon 13 Revertants/iig and were due to transversions of G to T or G to C. DNA adduci formation by carcinogenic heterocyclic amines CompoundIQMelQIQxMelQx4,8-DiMeIQx7,8-DiMeIQxPhIPCre-P-1Trp-P-1Trp-P-2Glu-P-1Glu-P-2Phe-P-lOrn-P-1AÂ«CMeAaCAflatoxin was detected by the i:P-posllabeling method (71-75). All het erocyclic amines examined except PhIP produced higher levels of DNA adducts in the liver than in other organs: with PhIP, which is not a hepatocarcinogen, the adduci level in the liver was lower than in the olher organs, including the lung, pancreas, and heart (74). Furthermore, the DNA adduci levels of various helerocyclic amines in ral liver correlaled well with the carcin ogenic potencies of these compounds in the liver. Thus, the levels of DNA adducls formed by carcinogenic doses of heler ocyclic amines in the liver are probably reliable indicators of their hepatocarcinogenicities. Some organs in which the heterocyclic amines form DNA adducts may develop cancers unless animals die from the first B,AF-2*4-Nitroquinoline dominant tumor. The presence of DNA adducts and histopalh- -oxideBenzo(a)pyreneMNNGf/V-Nitrosodieth>1 ological changes in the pancreas and salivary glands of rats treated with 0.08% MeAaC, which induces atrophy of these organs and is toxic to rats, suggest that lower doses of this lamineyV-NitrosodimethylamineTA98433.000661,00075,000145,000183,000163.0001,80019.00039.000104,20049.0001,9004156,8003002006.0006.5009703200.000.020.00TAIOO7,00030,0001,50014,0008,0009,9001204001,7001,8003,2001,200232012028.00042,0009.9006608700.150.23 compound can produce lumors in ihese organs (71). However, " See Table 1 for abbreviations. DNA from ihe kidney and heart of animals given helerocyclic * AF-2, 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide. amines conlains relaiively high levels of Ihe same kinds of DNA c MNNG: Ar-Methyl-A"-nitro-iV-nitrosoguanidine. 2094s

Table 3 Carcinogenicities of heterocyclic amines in rats and mice

ChemicalSpeciesIQ organsLiver, Rats small & large intestines, Zymbal gland, clitoral gland, skin MiceMelQ 0.030.03 Liver,lungLarge loirstoni, irk

RatsMiceMelQx intestine, Zymbal gland, skin, oral cavity, mammary gland 0.04.0.010.04 Liver,forestomachLiver, 44454647

Rats Zymbal gland, clitoral gland, skin MicePhIP 0.060.04 Liver,systemLarge lung, hematopoietic

Rats intestine, mammary gland MiceTrp-P-1 0.040.015 LymphoidtissueLiver 48495050515251 RatsMiceTrp-P-2 0.020.020.05 LiverLiverLiver, MiceGlu-P-1

Rats small & large intestines, Zymbal gland, clitoral gland MiceGlu-P-2 0.050.05 Liver,vesselsLiver, blood

RatsMiceAaC small & large intestines, Zymbal gland, clitoral gland 0.050.080.08Target Liver,vesselsLiver, blood 525252

MiceMeAaC vesselsLiver,blood

MiceConcentration(%)0.03 blood vesselsReference414243

Table 4 Amounts of heterocyclic amines in cooked foods" food)SampleBroiled Amount (ng/g cooked MelQx2.11 beef Fried ground beef 0.64 0.56 0.19 0.21 Broiled chicken 2.33 0.81 38.1 0.12Trp-P-20.250.18 Broiled mutton 1.01 0.670.10PhIP15.742.5 0.15AaC1.200.212.50MeAaC0.19 Food-grade beef extract 3.10 3.62 Fried cod fishIQ0.19 0.16MelQ 0.03 6.444,8-DiMeIQx0.12 69.2Trp-P-10.21 ' See Table 1 for abbreviations. is a relatively abundant heterocyclic amine in cooked food. The their carcinogenic dose, suggest that the carcinogenic effects of calculated average intake of carcinogenic heterocyclic amines is heterocyclic amines are additive or synergistic (86). This may about 0.4â€”16ng per day, per capita. Carcinogenic heterocyclic also be true for other environmental carcinogens. The total amines have been detected in all urine samples examined from exposure to more than 10 heterocyclic amines formed in cooked healthy volunteers eating normal diets but not in the urine of food could be, at most, several 100-fold less than the TD50 postoperative patients receiving parenteral alimentation (85). values of the individual carcinogenic heterocyclic amines (76). This indicates that humans are normally continuously exposed Painting a limited amount of Trp-P-2, which itself was not to heterocyclic amines in foods and that some level of exposure carcinogenic, on mouse skin resulted in skin tumor formation to heterocyclic amines is unavoidable. when the skin was treated with a tumor promoter, 12-O-tetra- The amounts of individual heterocyclic amines consumed decanoylphorbol-13-acetate (87). Similarly, a combination of a daily are too small to explain human carcinogenesis, assuming limited amount of IQ and a sufficient amount of phÃ©nobarbital that the susceptibility of humans to heterocyclic amines is the induced liver tumors in rats, although the same amount of IQ same as that of rodents. However, the results of medium-term alone did not induce tumors (88). A strain of Long-Evans rats experiments in which five heterocyclic amines were adminis with cinnamon-like coat color (LEC), which develops hepatitis tered simultaneously, each at one-fifth and one-twenty-fifth of spontaneously 5 months after birth and hepatomas 14 months after birth, is much more sensitive than normal Long-Evans rats to induction of hepatomas by MelQx.5 Such findings indicate that the carcinogenic potency of heterocyclic amines is readily modified by various factors. NH, NHOH NHOR < < < ,N-CH, P450IA2 N-CH, N-CH, Humans are continuously exposed to various mutagens-carcinogens, both autobiotic and xenobiotic. The multiple genetic N-Hydroxy-IQ R:-COCH, IQ-Guanme base adduct alterations present in human cancer cells could be produced by -so; many kinds of xenobiotic mutagens-carcinogens, each at a very

5H. Sone, M. Maeda, H. Kushida, K. Wakabayashi, K. Enomoto, M. Mori, Fig. 2. Metabolic activation pathway of IQ T. Sugimura, and M. Nagao, manuscript in preparation. 2095s

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1992 American Association for Cancer Research. FOOD-DERIVED MUTAGENS AND CARCINOGENS low level. In addition, autobiotic formation of oxidative agents 18 Nukaya, H., Watanabe, H.. Ishida, H., Tsuji, K., Suwa, Y., Wakabayashi, K., Nagao. M., Sugimura, T., and Kosuge, T. Isolation and structural in vivo and cell replication increase the chances of genetic determination of a mutagenic substance in creatine pyrolysate. Chem. IMiarm. alteration. Bull. (Tokyo), 39: 533-535, 1991. Total avoidance of exposure to heterocyclic amines is impos- 19 Ã–vervik,E., and Gustafsson, J-A. Cooked-food mutagens: current knowledge of formation and biological significance. Mutagenesis, 5: 437-446, 1990. sible, but reducing the exposure level as much as possible is 2Q Kasai, H., Yamaizumi, Z., Wakabayashi, K., Nagao, M., Sugimura, T., advisable. For example, charring of food during cooking should Yokoyama, S., Miyazawa, T., Spingarn, N. E., Weisburger, J. H., and be avoided. Direct contact of meat and fish with a naked gas Nishimura, S. Potent novel mutagens produced by broiling fish under normal flame or charcoal is also not recommended: a microwave oven conditions. Proc. Jpn. Acad.. 56B: 278-283, 1980. 21. Kasai, H., Yamaizumi, Z., Wakabayashi, K., Nagao, M., Sugimura, T., may be preferable. Cooking meat and fish in aluminum foil Yokoyama, S., Miyazawa, T., and Nishimura, S. Structure and chemical would reduce charring, and a simple method for removing synthesis of Me-IQ, a potent mutagen isolated from broiled fish. Chem. heterocyclic amines is mechanical separation of charred parts Lett., 1391-1394, 1980. 22. Becher, G., Knize, M. G., NÃ©s,I. F., and Felton, J. S. Isolation and of broiled fish and meat from edible portions. Soybean protein identification of mutagens from a fried Norwegian meat product. Carcino suppresses mutagen formation in fried hamburger (89). 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Keiji Wakabayashi, Minako Nagao, Hiroyasu Esumi, et al.

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