Carcinogens and Mutagens Present as NaturalComponents of Food or Induced by Cooking

Michael J. Privai

IntroductionThis paper reviews the available data on mutagens and carcinogens appearing in our food,

not as additives, pesticide residues, or contaminants, but rather as natural components of foodor as a result of cooking. The remarks are confined to mutagens and carcinogens that arelikely to be found in typical US diets; such carcinogens as bracken fern, pyrrolizidine alkaloids,and cycasin are not discussed, since they are not known to be present to any significant degreein the food supply in this country. Reference is made to both mutagenesis and carcinogenesis.Mutagenicity is the capacity to cause changes in the genetic material that can be passed onto future generations of cells or organisms. Mutagenicity tests are often used to screen chemicalsfor potential carcinogenicity.

The most widely used test for mutagenesis is the Ames test[l,2], which utilizes Salmonellatyphimurium bacteria as target cells. Since DNA is chemically similar in all living organisms,bacteria are considered to be acceptable target organisms for genetic tests. Chemicals thatreact with and cause mutations in bacterial DNA are also likely to cause mutations inmammalian DNA.

The Ames mutagenicity test is also the most widely used screening test for chemical car-cinogens. The basic reason that the Ames test and other mutagenicity tests are accepted asprescreens for carcinogenicity is that a number of investigators have concluded that there isreasonably good correlation between mutagenicity and carcinogenicity[3-7]. The actual percentcorrelation depends greatly on the selection of chemicals used. Most of the mutagenicity datathat will be discussed here are from the Ames Salmonella typhimurium assay.

Mutagens and Carcinogens Resulting From Cooking

About 20 years ago, two groups of investigators, Seppilli and Scassellati Sforzolini[8] andLijinski and Shubik[9], reported that meat cooked on a grill over charcoal or a gas flamecontained polynuclear aromatic hydrocarbons, including carcinogens such as benzo(o)pyrene.Subsequent experiments by Lijinski and Ross[10] confirmed the hypothesis that the productionof polynuclear aromatic hydrocarbons during charcoal broiling is dependent upon the fatcontent of the meat. They concluded that the source of the hydrocarbons was the drippingfat being pyrolyzed by the hot charcoal, and they recommended procedures for reducing the

The author is affiliated with the Genetic Toxicology Branch, Center for Food Safety andApplied Nutrition, US Food and Drug Administration, Washington, DC 20204.

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level of such hydrocarbons. These included avoiding contact of food with the flame, using apan to prevent dripping fat from falling into the flame, and using meat with less fat.

In 1977, Nagao et al.[ll] reported that benzo(a)pyrene and similar hydrocarbons accountfor only a small fraction of the total mutagenic activity of cooked meat or fish when thesurfaces of the meat or fish are charred. They extracted the charred surfaces of beef and fishwith dimethylsulfoxide and tested the mutagenicity of the extracts. They found that themutagenic activity was equivalent to 4,500 ¡xg of benzo(c)pyrene per kilogram of meat or fish.(Lijinski and Shubik[9] had reported that charcoal broiled beef contained no more than 8 jugof benzo(a)pyrene per kilogram of beef.)

Nagao and co-workers then began to search for the source of the mutagenic activity anddiscovered that the smoke from pyrolyzed protein was highly mutagenic, while smoke con-densates from pyrolyzed DNA, RNA, starch, or vegetable oil were only slightly mutagenic[12].When each individual amino acid was pyrolyzed, the pyrolysate of tryptophan was found tobe the most mutagenic, but almost all of the common amino acids gave rise to mutagens whenpyrolyzed[13,14].

The principal mutagenic components of some of the amino-acid pyrolysates have beenpurified and characterized chemically[15], and have been designated Trp-P-1, for TryptophanPyrolysis 1, and so on.* Figure 1 shows two isolates from pyrolyzed tryptophan, two fromglutamate, one from lysine, and one from phenylalanine. All of the potent mutagens that havebeen isolated from pyrolyzed amino acids or cooked foods so far are heterocyclic amines,except for Lys-P-1. All of those tested for carcinogenic activity so far appear to be carcinogenicwhen fed to mammals.

As mentioned earlier, the first reports of these potent mutagens resulted from mutagenicitytesting of the charred portions of meat and fish. Obviously, the foods were exposed to veryhigh temperatures. The mutagens were isolated from amino acids pyrolyzed at temperaturesexceeding 250°C. However, it is now known that mutagens are produced when meat is heatedto much lower temperatures. This fact was discovered by Commoner and his colleagues[16,17]who showed that mutagenic activity appears during the boiling of beef extract, thus demon-strating that mutagens are formed when beef is heated to only about 100°C.

In the Ames test[l,2], chemicals are routinely tested both in the presence and absence ofan in vitro metabolic activation system derived from rodent liver and referred to as S9 mix.Commoner had been using the Ames test to evaluate a large number of chemicals. Duringthis work, he noted that when the bacterial strains were plated in the absence of the testchemical but presence of the S9 metabolic activation system, the spontaneous mutant countswere usually higher than those obtained in the absence of the S9 mix. Before Commoner'swork, those of us working with the Ames assay had generally assumed that this increase wasdue to the presence of histidine in the S9, which would enable more cells to grow on the plateand thus give rise to more spontaneous mutants. Commoner had a different explanation: Hespeculated that either there was a mutagen in the S9 or there was a mutagen somewhere elsein the system that required S9 for activation.

Commoner's group then discovered that the beef extract in the nutrient broth in which thebacterial cells were grown before the test had mutagenic activity. The beef extract is preparedby boiling a beef broth down to 20% or less of its original volume. By showing that themutagenic activity appears during the boiling step, Commoner's group demonstrated thatmutagens are formed when beef is heated to only about 100°C. The discovery of the mutagenicity

*Abbreviations used in this paper: Trp-P-1, 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole; Trp-P-2,3-amino-l-methyl-5H-pyrido[4,3-6]indole; Glu-P-1, 2-amino-6-methyldipyrido[l,2-a:3',2'-d]imidazole;Glu-P-2, 2-aminodipyrido[1,2-a:3',2'-d]imidazole; Lys-P-1, 3,4-cyclopentenopyrido[3,2-a]carbazole;Phe-P-1, 2-amino-5-phenylpyridine; IQ, 2-amino-3-methylimidazo[4,5-f]quinoline; MeIQ, 2-amino-3,4-dimethylimidazo[4,5-f]quinoline; MeIQx, 2-amino-3,8-dimethylimidazo[4,5-f]qumoxaline.

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Trp-P-2

Glu-P-2

N-CH3

Figure 1. Structures of mutagens isolated from pyr-olyzed amino acids and cooked foods, (Abbreviationsdefined in text.)

MelQx

of beef extract led Commoner's group to the finding that hamburgers as normally cooked aremutagenic.

Three mutagens that are not amino-acid pyrolysis products have been reported to be presentin beef extract (Figure 1): IQ, MelQ, and MeIQx[18]; however, there is some controversy asto whether MelQ is present[19]. The major mutagen that has been identified in fried groundbeef is MeIQx[18,20,21], although many unidentified mutagens are also present[21]. Someinvestigators[20-22], but not others[23], have identified IQ as one of the mutagens in friedground beef. IQ and MelQ were originally identified as mutagens isolated from broiledfish[24,25]; MelQx was first isolated from fried beef[26]. In addition, work is in progress inseveral laboratories to purify and identify additional unknown mutagens from cooked meatand from beef extract.

At the time they were discovered, MelQ, IQ, MelQx, and some of the amino-acid pyrolysisproducts were the most potent mutagens known when tested in Salmonella typhitnurium[2T\.More recently, some nitropyrenes have been tested that have even exceeded the mutagenicpotency of these cooking-induced mutagens[28]. It is important to understand, however, that

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a chemical that is a potent mutagen in the Ames test is not necessarily a potent carcinogenin mammals.

Unfortunately, data on the carcinogenicity of MelQx are not yet available. Although dataon the carcinogenicity of several of the cooking-induced mutagens fed to animals have nowbeen published, these data do not include dose-response information[29-33].

It is, however, still interesting to compare the dose of, for example, Trp-P-1, reported tobe carcinogenic in a published experimental], with the dose to which humans may be exposedin the eating of cooked meat. Trp-P-1 at 200 ppm in the diet induced hepatocellular tumorsin 21% and 62% of male and female mice, respectively, with the combined overall responsefor both sexes being 42%[31]. Yamaguchi et al.[34] found that the mutagenicity of Trp-P-1extracted from very well done broiled beef corresponded to about 53 ng of Trp-P-1 per gramof beef, or 53 ppb. If we assume, as the authors do, that the overall extraction was about 50%efficient, then the original concentration of Trp-P-1 would be 106 ng/g cooked beef. Theaverage per capita consumption of meat and meat products in the US has been reported tobe 188 g/day[35]. If it all contained the level of Trp-P-1 found in very well done beef, thenthe average daily human dose of Trp-P-1 for a 70-kg person would be about (106 ng/g) X(188 g/day)/70 kg, or 285 ng/kg/day. The dose of Trp-P-1 in the carcinogenesis study onmice[31] was 0.53 mg/day for male mice and 0.45 mg/day for female mice, which comes toabout 15 mg/kg/day, assuming that the average male mouse and female mouse weighedabout 35 g and 30 g, respectively. Thus, the difference between the daily human dose frombeef and the daily mouse dose in this experiment would be a factor of about 50,000.

This factor of 50,000 can be viewed in different ways. We could say that the difference inthe possible human dose and the level that caused tumors in the animals is so large that thereappears to be little reason for concern. Also, the calculation was based on the assumption thatall the meat consumed in the US contains as much Trp-P-1 as very well done beef, whichcertainly leads to a great overestimate of Trp-P-1 intake. The possibility also exists that humanconsumption of cooked foods over the millenia has led to the evolution of detoxificationmechanisms that are not present in laboratory animals, whose ancestors did not generally eatcooked foods; that could mean that we are, in fact, much less sensitive than these animals tothe carcinogenic effects of cooking-induced mutagens. However, that is not a very convincingargument because cancer generally occurs in people beyond their child-bearing and child-rearing years, and susceptibility to the carcinogenicity of cooking-induced carcinogens willtherefore have little effect on the process of evolution. That is, there may be little or no naturalselection against susceptibility to cancer from cooking-induced carcinogens.

On the other hand, we might look at this factor of 50,000 with some alarm. After all, Trp-P-1 is but one of many cooking-induced mutagens found in food. Many of these mutagenshave been reported to be carcinogens in mammals, although the actual carcinogenicity datafor only a few of them have so far been published. Trp-P-1 appears to account for perhapsonly 6% of the mutagenic activity of cooked beef[34; see also 19, 36], and it is reasonable toassume that the compound accounts for only a small fraction of the total carcinogenic riskfrom cooking-induced mutagens.

The human risk of cancer from a chemical that is carcinogenic in animals can be estimatedby using mathematical models to extrapolate carcinogenic risk from high experimental dosesto low human exposure doses. One simple, conservative model for estimating carcinogenicrisk at low doses is the "one-hit" model, which states that

P = 1 - e**

where P is the probability (risk) of a tumor, d is the dose of the carcinogen, and k is a constant

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to be determined experimentally. According to this model, risk is proportional to dose at lowdoses, while at high doses the risk approaches 100% asymptotically.

For Trp-P-1 in the experiment of Matsukura et al.[31], d = 15 mg/kg/day and P = 0.42;so we calculate that k = 0.0363 (mg/kg/day)"1. At the estimated human dose of 285 ng/kg/day, the risk of cancer from Trp-P-1 would be calculated as about one per 100,000 peopleover their lifetimes. Although that is a very small risk for any individual to incur, it wouldimply that Trp-P-1 alone could induce cancer in more than 2,000 of the Americans currentlyah've if such conservative risk extrapolations were accurate rather than resulting in an over-estimate of risk. We do not now have sufficient data to estimate the risk for all cooking-induced mutagens.

Mutagens and Carcinogens Naturally Occurring in Foods

Flavonoids

Although cooking-induced mutagens appear to be present in minute quantities in our dailydiets, there are some mutagens that we all consume at fairly high levels: the mutagenicflavonoids. These fall into several chemical categories: the flavones, such as wogonin, nor-wogonin, isowogonin[37], apigenin, chrysoeriol, and pedalitin[38]; the flavanones, such as 7,4'-dihydroxyflavanone[38]; the flavanonols, such as taxifolin[38,39]; and the flavonols. Of these,only some of the flavonols have been extensively investigated for mutagenicity beyond testingin bacteria or for carcinogenic activity.

Flavonols are derivatives of flavonol, which is 3-hydroxyflavone (Figure 2). Flavonols arewidely distributed in plants, including many of those we eat as food. The most commonlyfound flavonol is quercetin; another widespread flavonol is kaempferol. Usually, but not always,flavonols are present in plants in the form of glycosides. Rutin is a common glycoside ofquercetin; astragalin is a glucoside of kaempferol.

Many flavonols, including quercetin and kaempferol, are mutagenic to bacteria[38-42].Although quercetin is a direct-acting mutagen, its activity is enhanced considerably by theaddition of the in vitro metabolic activation system containing rat liver S9. Kaempferol, onthe other hand, is mutagenic only in the presence of S9.

As previously stated, flavonols usually exist in plants as glycosides, bound to various sugarmolecules. Hydrolysis of glycosides can occur in the lower bowel and will result in the releaseof the free mutagenic flavonol. The metabolic activation system of the standard Ames testcan mimic many of the reactions that occur in the liver, but it is not efficient at performingsome of the reactions that occur primarily in the gut, such as hydrolysis of glycosides. Toovercome this shortcoming, various modifications of the mutagenesis assay have been made.For example, extracts containing mixed glycosidases have been added to the reaction mixture.In the presence of such enzyme-rich extracts derived from the mold Aspergillus niger[38], thesnail Helix pomatia[39], the cecal contents of rats[39], and human feces[43], rutin and otherflavonol glycosides have been found to be mutagenic when tested in S. typhimurium.

Although results of the bacterial mutagenicity test raise the concern that widely consumedflavonols such as quercetin and kaempferol may be carcinogens, this test is by no meansdefinitive. A variety of genetic assays in more complex systems, as well as mammalian celltransformation assays, can contribute to the safety evaluation of substances identified asbacterial mutagens. Of course, the chronic whole animal carcinogenesis assay is consideredthe definitive test, but it is expensive and time-consuming to perform and it is often helpfulto confirm the results with a variety of in vitro assays.

Unfortunately, the overall safety evaluation picture for quercetin is somewhat unclear, asshown in Table 1. For example, some mutagenesis tests in cultured mammalian cells havebeen positive and others negative. Carver et al. [48] and van der Hoeven et al. [45] have suggestedthat the apparent discrepancies may be explained by assuming that quercetin is capable of

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OH

OH OKaempferol

O-GluII

OH ORutin Astragafin

Figure 2. Structures of flavonol, two flavonol derivatives, and two glycosides of flavonol derivatives.

causing DNA breaks and chromosomal aberrations, but not point mutations, in mammaliancells. This explanation assumes that the positive result in V79 cells at the HGPRT locusreported by Maruta et al. [44] was due to an artifact resulting from the technique used for theexperiment[45,48]. It is currently thought that positive results in the thymidine kinase locusof mouse lymphoma cells may result from either chromosome aberrations or point muta-tions[60], and the same may be true of thymidine kinase mutations in Chinese hamster ovary(CHO) cells. Thus, quercetin may act to produce thymidine kinase mutants by inducing onlychromosomal aberrations, which would explain why the other mutagenesis assays in mam-malian cells in culture are negative: those loci can only respond to point mutagens. The sex-linked recessive lethal test in Drosophila melanogaster responds to both chromosomal aber-rations and point mutations; the micronucleus test is an assay for chromosomal aberrations.

Thus, all of the mutational results obtained in mammalian cells, and even in Drosophila,could be explained on the basis of chromosomal aberrations rather than point mutations. That

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Table 1. Summary of Representative Mutagenicity and Carcinogenicity TestResults for Quercetin

Test system

BacteriaSalmonella typhimurium

Mammalian cell cultureChinese hamster (V79)Chinese hamster (V79)Mouse lymphoma (L5178Y)Chinese hamster ovary cellsChinese hamster ovary cellsChinese hamster ovary cellsChinese hamster ovary cellsChinese hamster ovary cellsChinese hamster fibroblastsHuman fibroblastsHuman lymphocytes

Whole animalsDrosophila melanogasterMouse bone marrowMouse bone marrow

Transformation in culturedmammalian cells

Hamster embryo cellsBalb/c 3T3 cells

Carcinogenicity in animalsRatRatMouseHamster

a: Abbreviations for genetic endpoints:

Endpoint"

Mutagenicity (Ais)

Mutagenicity (HGPRT)Mutagenicity (HGPRT)Mutagenicity (TK)Mutagenicity (TK)Mutagenicity (APRT)Mutagenicity (HGPRT)Mutagenicity (ATPase)Chromosome aberrationsChromosome aberrationsChromosome aberrationsChromosome aberrations

Mutagenicity (SLRL)MicronucleiMicronuclei

Altered coloniesFocus formation

TumorsTumorsTumorsTumors

his, histidine biosynthesis;phoribosyl transferase; TK, thymidine kinase; APRT, adenineNa+/K+-ATPase; SLRL, sex-linked recessive lethals.

Result Reference

+ [38-42]

+ [44][45]

+ [45-47]+ [48]

[48][48][48]

+ [48,49]+ [50]+ [50]+ [50]

+ [51]+ [52]

[53]

+ [54]+ [47]

+ [55,56][57][58][59]

ÍGPRT, hypoxanthine-guanine phos-phosphoribosyl transferase; ATPase,

conjecture, however, is difficult to reconcile with the fact that quercetin is mutagenic in bacteria,which can only respond to chemicals that induce point mutations, since bacteria do not havechromosomes. Quercetin may act by quite different mechanisms in bacteria and in animalcells, which may be why the presence of S9 greatly stimulates the mutagenicity of quercetinin bacteria but actually reduces its mutagenicity in mouse lymphoma cells[45,47]. These findingsmay be consistent with the idea that quercetin causes point mutations in bacteria but can onlycause chromosomal aberrations, rather than point mutations, in mammalian cells.

The situation becomes even more confusing when the ability of quercetin to transformmammalian cells and induce tumors is considered. Quercetin appears to be capable of trans-forming both early passage hamster embryo cells[54] and Balb 3T3 cells[47]. Such positiveresults in cell transformation assays would probably be considered to be the strongest availablein vitro evidence for carcinogenicity.

When the in vivo results in chronic feeding studies are considered, further apparent incon-sistencies are found. Pamukcu et al.[55] fed noninbred albino rats a diet containing 0.1%quercetin for 58 weeks and found intestinal tumors in 80% of the animals and bladder tumorsin 20%, compared with none in controls. The intestinal tumors were in the ileal segment of

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the small intestine. Of the 20 affected rats, 4 had adenomas, 7 had fibroadenomas, and 9 hadadenocarcinomas. All five of the urinary bladder tumors were transitional cell carcinomas.Mesenteric lymphatic métastases were observed in three of the rats. Thus, from this experiment,the carcinogenicity of quercetin in rats seems to be clear.

However, when Hirono et al.[57] fed quercetin to inbred ACI rats at doses of 1% or 5%for up to 121 weeks, no significant increase in tumor incidence occurred. The doses in theirexperiments were 10 to 50 times the levels administered by Pamukcu et al.[55], and theduration of exposure was twice as long. The different results could possibly be due to the factthat Pamukcu et al., in Madison, WI, used noninbred Norwegian rats, while Hirono et al.,in Tokyo, used inbred ACI rats. The negative results in the ACI rats could not be attributedto poor absorption of quercetin from, or microbial degradation of quercetin in, the digestivetract[61]. In a recent experiment performed by the Wisconsin group[56], quercetin was foundto induce hepatomas in Sprague-Dawley rats and hepatomas and biliary adenomas in Fischer344 rats, but no significant increase in malignant tumors was reported. Thus, the ability ofquercetin to induce tumors in rats originally reported by Pamucku et al.[55] has been repro-duced in two additional rat strains, but the induction of malignant tumors and métastases wasapparently not repeated. It would seem to be quite important to determine the cause of theconflicting carcinogenicity results, considering the fact that we all ingest quercetin and itsglycosides each day. Additional experiments, conducted by the group in Tokyo, in which highlevels of quercetin were fed to ddY mice[58] or to golden hamsters[59] failed to demonstratethe carcinogenicity of quercetin.

Thus, the in vitro mutagenicity and transformation assays on quercetin give an array ofresults that is difficult to interpret. They do not indicate definitively whether or not quercetinis a carcinogen. In addition, the so-called definitive in vivo assays are contradictory.

The argument could be made that low-dose exposures of quercetin are very unlikely topresent a carcinogenic risk because the carcinogenic activity of quercetin reported by Pamukcuhas not been reproduced in other laboratories. On the other hand, it is difficult to ignore allof the positive mutagenicity, cell transformation, and carcinogenicity data on quercetin. Thefact that this compound caused point mutations in bacteria raises the possibility that negativeresults in several mammalian point mutation tests may simply reflect differences in sensitivityamong those tests. In addition, we must remember that in the attempts of other investigatorsto repeat the positive carcinogenicity results of Pamukcu et al. [5 5], different types of animalswere utilized. The fact that quercetin has now been reported to induce tumors in three differentstrains of rats[55,56] would seem to tip the balance of the evidence in favor of a conclusionthat quercetin is carcinogenic.

In evaluating the potential health implications of consuming chemical carcinogens, it isessential to have some notion of the differences between those doses that have caused tumorsin experimental animals and those to which humans are exposed. Flavonol glycosides, par-ticularly quercetin and kaempferol glycosides, are found in the edible portion of the majorityof food plants, including citrus and other fruits, berries, leafy vegetables, roots, tubers, andbulbs, herbs and spices, legumes, cereal grains, tea, and cocoa[62]. I have not been able tofind any estimates of the average daily human level of intake of quercetin, but in a recentreview, Brown[62] estimated daily intake of all mutagenic flavonol glycosides to be about 50mg/day. If all the mutagenic flavonols consumed (50 mg/day = 0.71 mg/kg/day) areconsidered to be as active as the quercetin used in the study of Pamukcu et al.[55], in which0.1% in the diet (about 50 mg/kg/day) was carcinogenic to rats, then the difference betweenthe carcinogenic dose in rats and the estimated human dose is only a factor of about 70. Thatis a very small margin of safety between the level reported carcinogenic and the level we mayconsume. It would work out to a very high lifetime risk estimate, exceeding one cancer per50 exposed people, if the conservative one-hit model is used. Even if only quercetin itself is

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considered, the risk estimate would exceed one in 500, assuming that it constitutes at least10% of the mutagenic flavonols we consume.

If we became convinced that these common constituents of many of our foods were car-cinogenic and that the risks were high, there are several strategies that could be employed.One strategy might be to recommend that people alter their diets to consume fruits andvegetables that contain lower levels of carcinogenic flavonols, but that would have othernutritional consequences that would have to be evaluated. Another approach might be toattempt to breed plants that had lower levels of the chemicals.

It is not at all clear whether elimination of flavonols from our diet would be totally beneficialto human health. Flavonols such as quercetin and kaempferol are antioxidants, mostly becauseof their phenolic hydroxyl groups. As antioxidants, they may help to protect foods againstoxidative deterioration. For example, foods that are very rich in flavonoids, such as onionsand black currants, have unusually stable vitamin C contents[63]. There have also been claimsthat flavonoids can enhance the potency of ingested vitamin C, perhaps by stabilizing it inthe body, although those claims do not appear to be well documented^]. However, naturalvitamin C tablets sold in health food stores are supplemented with so-called bioflavonoids,including rutin, presumably for the purpose of enhancing the potency of the vitamin.

There have also been numerous therapeutic claims for flavonoids, especially rutin. In fact,rutin was formerly an official drug in the US and has been used in treating capillary hemorrhagedue to increased capillary fragility in degenerative vascular diseases (e.g., arteriosclerosis andhypertension), diabetes, and allergic manifestations[64]. Rutin's reputed ability to decreasecapillary permeability and fragility has led some to refer to it as "vitamin P" or the "perme-ability vitamin."

Although the therapeutic claims appear to be inadequately documented, flavonoids, espe-cially rutin, are available in health food stores as food supplements and are presumablyconsumed because of the claimed health effects. If mutagenic flavonols and their glycosidesor other flavonoids were conclusively demonstrated to be carcinogens, then a balanced judgmentas to the appropriate course of action would require a careful assessment of the possible healthbenefits as well as the health risks associated with these natural components of food.

HydrazinesOver the past several years, Toth[65] has been studying the carcinogenicity of various

hydrazine-type compounds, including some that are found in edible mushrooms. Much of hiswork has focused on carcinogens present in Gyromitra esculenta, one of the false morelmushrooms. This mushroom must be picked in the wild, because it is not sold commerciallyin the US. For that reason, and also because of its known toxicity, it is consumed by relativelyfew Americans. Of much greater interest is Toth's work on carcinogens in the commoncommercial edible mushroom, Agaricus bisporus. This mushroom contains about 400 ppm ofagaritine, which is L-glutamic acid 5-[2-[4-(hydroxymethyl)phenyl]hydrazide][66]. Althoughagaritine itself does not appear to be carcinogenic, at least to the extent that it has beentested[67], it apparently can be metabolized by two separate enzymatic pathways present inmushrooms to 4-(hydroxymethylbenzenediazonium) ion (HMBD)[68]. HMBD has been re-ported to be present in raw mushrooms at a level of 0.6 ppm; however, it is unstable and isprobably destroyed by cooking.

When Toth et al.[69] prepared HMBD stabilized as the tetrafluoroborate salt, they foundthat a single intragastric administration of 400 mg/kg to Swiss albino mice resulted in a 31%incidence of glandular stomach tumors. Of the mice with these tumors, 61% developedadenocarcinomas and the remaining 39% only adenomas. No tumors of the glandular stomachwere seen in control animals.

In comparing the dose given to the animals with the levels of HMBD to which humansmight be exposed, we are faced with a problem: the mice were given only a single administration

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HpC—CH — CH2

Safrole

OCH, OCH,

H2C—CH = CHo

Estragóle

OCH,

H2C—CH = CH2

Methyleugenol

OCH,

CH3Ö

OCH,

CH = CH-CH3

Isosafrole/?-Asarone

Figure 3. Structures of some carcinogenic alkenylbenzenes found in foods.

of 400 mg/kg, but our consumption of mushrooms is spread over time. We can compare thedose given to the mice to the total amount that a person might consume over a 10-year period,for example, but we cannot predict whether the low-level chronic exposure will be more orless hazardous than the high single dose. Per capita consumption of fresh mushrooms in theUS is approximately 1.5 pounds (0.68 kg) per year[70]. If all of these mushrooms were consumedraw and contained 0.6 ppm (0.6 mg/kg) of HMBD, then in 10 years the average 70-kg personwould consume a dose of about (0.68 kg/year) X (10 years) X (0.6 mg/kg)/(70 kg) =0.058 mg/kg of HMBD, or about 1/7,000 the dose that caused stomach tumors in 31% ofthe mice. This results in an extrapolated lifetime cancer risk for HMBD in mushrooms of 5X 10"5 or one in every 20,000 people. However, since most of the mushrooms consumed areactually cooked before being eaten, the calculated risk from HMBD should be far less thanthat figure. For example, if only 1% of the mushrooms are eaten raw, then the extrapolatedrisk is less than one per million.

Toth et al. [69] have suggested that agaritine, which is present at much higher levels inmushrooms than is HMBD, may be converted to HMBD by human metabolism. If that turnsout to be true, then the estimated risk associated with eating mushrooms will be much greaterthan that for preformed HMBD in the mushrooms.

Alkenylbenzenes

Herbs and spices contain numerous chemicals that give them their characteristic flavorsand odors. Figure 3 shows a series of carcinogenic alkenylbenzene compounds found in spices.

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Safrole is the principal ingredient in oil of sassafras, constituting 80-90% of the oil[64].Safrole was found to be carcinogenic over 20 years ago[71]. At one time, oil of sassafras wasthe principal flavoring ingredient in commercial root beer. As a result of the finding thatsafrole is a carcinogen, neither safrole nor oil of sassafras can be legally added to food, exceptthat extract of sassafras can be used if the safrole has been removed[71-73]. Commerciallyavailable root beer is now flavored without safrole, but sassafras tea is still available in healthfood stores. Some of the tea is sold as a safrole-free liquid concentrate; some is sold as "sassafrasbark" and labeled to indicate that it is not for use as a food (although I was clearly told bya salesperson in a health food store that this sassafras bark was meant to be used to preparea tea); and some is sold as sassafras bark and clearly labeled as a tea.

Estragóle (also called methylchavicol) is found at high concentrations in both sweet basiland tarragon. Since Americans apparently consume 15-20 times as much basil as tarragon,sweet basil appears to be our principal dietary source of estragóle. The best estimate I havebeen able to make, based on importation and estimated production figures[74], is that theaverage American consumes about 30 mg of sweet basil per day. Estragóle constitutes up toabout 25% of the volatile oils of the spice[75]. Since basil yields about 1% of its weight asvolatile oil[64], we can calculate that a 70-kg person consuming 30 mg of basil daily will beconsuming about 1 /ig of estragole/kg/day. In one of the carcinogenesis experiments onestragóle performed by Miller et al.[76], female CD-I mice were fed diets containing 0.23%(2,300 ppm = about 345 mg/kg/day) estragóle for 12 months. At the end of 20 months,hepatomas were found in 56% of the animals, compared with none in controls. The estimateddifference between the human dose of estragóle and the dose that caused tumors in the micewould be a factor of 345,000. The extrapolated lifetime carcinogenic risk would be 2.4 X 10"*,or one cancer per 420,000 people.

Methyleugenol constitutes about 4% of the weight of sweet bay oil[64]. This chemical,which is closely related to estragóle and safrole, induced liver tumors in mice when administeredintraperitoneally before weaning[76]. Since no data are available on the importation or pro-duction levels of sweet bay or its oil or on the carcinogenicity of orally administered meth-yleugenol, it is not possible to estimate the level of human consumption of the chemical orthe possible risk associated with its ingestion.

/?-Asarone is the principal component of oil of calamus[64]. A sample of oil of calamuscontaining 75.8% /3-asarone produced tumors of the small intestine when fed to rats[77]. Asa result of this finding, oil of calamus was banned from use in food in the US[78,79].

Isosafrole, a minor component of ylang-ylang oil[64], induced liver tumors when fed to ratsor mice[80]. Although this oil is approved for use as a "generally recognized as safe" ingredientin food[81], its use in foods appears to be at very low levels, generally below 0.001%, in somebeverages, desserts, and baked goods[64]. In 1983, 32,000 kg of ylang-ylang oil were importedinto the US[82], or about 100 mg/person. Since much of the ylang-ylang oil imported is usedas a fragrance in soaps, cosmetics, and perfumes, and since isosafrole is only a minor componentof the oil, it would seem that exposure to isosafrole in foods is very low, but it is not possibleto estimate with available data.

Other Carcinogens in Foods

An alcohol extract of ground black pepper has been reported to be carcinogenic whenapplied to the skin of mice[83]; however, the experiment yielding this result was poorlyconducted, as evidenced by the fact that many animals died early in the experiment becauseof respiratory infections and fighting. Since significant numbers of tumors were seen in treatedanimals at various sites away from the site of application, and since we do consume largeamounts of black pepper, the carcinogenicity of that condiment may deserve further investi-gation.

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Seeds of brown or black mustard contain sinigrin (potassium myronate) as well as theenzyme myrosin (myrosinase), which can hydrolyze the sinigrin to yield allyl isothiocyanate(oil of mustard)[64]. This oil of mustard is considered to be "generally recognized as safe"for use in foods by the US Food and Drug Administration^ 1]. (The oil of white or yellowmustard, which is the most common type of mustard generally used, is a different compound,/>-hydroxybenzylisothiocyanate[64,84].) In a recently published carcinogenesis study, allyl iso-thiocyanate induced transitional cell papillomas of the urinary bladder when administered bygavage to male rats at doses of 12 or 25 mg/kg, five times per week for 103 weeks[85,86].

Daily consumption of brown mustard in the US has been estimated to be 18 mg/day[87].If it is assumed that all of the sinigrin in that mustard is converted to allyl isothiocyanateand that the yield of allyl isothiocyanate from the mustard is 0.9%[64], then the average dailydose of allyl isothiocyate from mustard would be 0.16 mg/day, or about 0.0023 mg/kg/day.The average daily intake of allyl isothiocyanate added to food as an oil has been estimated tobe about 0.2 mg/day[87]; most of it is synthetic, rather than derived from mustard seeds.Thus, the dose of allyl isothiocyanate that induced benign bladder tumors in rats was only3,700 times the estimated average human dose from mustard and only 1,700 times the totaldose from natural and synthetic sources combined. These are relatively low safety factors fora carcinogen.

The compounds 5-methoxypsoralen (5-MOP) and 8-methoxypsoralen (8-MÖP) are foundin certain foods, including parsnip root[88], figs[89], and parsley[89]. Psoralens are toxic, butonly when the treated individual is exposed to ultraviolet light; this effect is known as pho-tochemical toxicity. The toxic effects of psoralens from diseased celery on celery workers havebeen known for many years[90-92], and the compounds have recently been identified inhealthy celery as well[93]. In addition, oil of bergamot, which is considered to be "generallyrecognized as safe" for use in foods by the Food and Drug Administration[81], contains about0.30-0.36% 5-MOP[94]. It is used in candy and desserts[64] and also in some tea.

Both 5-MOP and 8-MOP are photocarcinogens; that is, they induce skin cancer in animalswhen skin application is followed by irradiation with ultraviolet light[95—97]. Intraperitonealadministration of 8-MOP followed by irradiation with ultraviolet light also results in tumorson the skin of mice[95,98]. Oral administration of 8-MOP was less effective than intraperitonealinjection, with several reports noting little or no carcinogenic effect[95, 98-101], but thephototoxicity of orally adminitstered 8-MOP and 5-MOP, and direct chemical analyses[102],demonstrate that the compounds do reach the skin following ingestion. A recent epidemiologicalstudy[103] on humans exposed to therapeutic oral doses of 8-MOP and to ultraviolet radiationfor the treatment of psoriasis has demonstrated a carcinogenic effect. Thus, the possibilityshould be considered that the presence of psoralens in food in conjunction with exposure tosunlight might increase the risk of skin cancer.

ConclusionA thorough analysis of the carcinogenic risks associated with cooking-induced and natural

constituents of foods is important for two reasons. First, it may be possible for us to eliminateor greatly reduce consumption of particular substances that are likely to be carcinogenic, ashas been done with safrole in root beer. Second, a realistic analysis of the natural and cooking-induced carcinogenic risks of foods will enable us to put in perspective the risks associatedwith added chemicals in our food.

The well-known Delaney clause of the Federal Food, Drug, and Cosmetic Act prohibitsthe use of food additives that have been shown to be carcinogenic upon ingestion by humansor animals. Federal regulatory agencies (the Food and Drug Administration and the Envi-ronmental Protection Agency) have been attempting for many years to define methods forestablishing permissible limits for residues in foods of carcinogenic chemicals that are nottechnically "food additives" covered by the Delaney prohibition—such as pesticides and

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contaminants. It is clear that carcinogens are numerous enough in our environment that theycannot all be avoided. Thus, the formulation of rational policies for the control of carcinogenswill require, at least in some cases, that we attempt to estimate the risks associated withexposures to these chemicals.

The evaluation of carcinogenic risk is fraught with uncertainties due to the necessity ofextrapolating data from animals to humans and from high to low doses, using mathematicalmodels of unknown validity. The inadequacy of almost all carcinogenicity data, especially thelack of dose-response information, adds to the problem.

It is interesting to try to compare the risks associated with exposure to carcinogenic com-ponents of food with the risks from deliberately added substances (natural or synthetic). Aconservatively calculated lifetime risk of less than 10~6 has been suggested for exposure toconstituents of substances added to food. The possible carcinogenic risks associated with anumber of the naturally occurring components of food may greatly exceed that level. However,the uncertainties discussed concerning each of those components makes it difficult to estimatethe risks.

The widespread exposure in the press given to Ames' recent review of naturally occurringand cooking-induced mutagens and carcinogens in food[104] and the commentary it engen-dered[105-107] indicate the significance that the discovery of these compounds may have inhelping shape public attitudes toward environmental carcinogens in general. Society mayeventually decide to accept risks from synthetic chemicals as long as the estimated risks aresmall compared with those resulting from cooking-induced or naturally occurring constituentsof food.

On the other hand, people might take the position that any additional carcinogenic risksthat can be avoided without undue hardship are unacceptable, even though we accept muchlarger risks from natural. constituents in our food. In fact, it could be argued that a highbackground of naturally occurring carcinogens is likely to make exposures to additional lowlevels of synthetic carcinogens even more dangerous than they would be by themselves. InFigure 4, for example, the low-dose portion of a possible dose-response curve for a carcinogenis plotted. If a dose, D, of a single carcinogen is given to a population, a small response, R,will be obtained. However, if the population is exposed to a background, B', of carcinogensoperating by the same mechanism as the carcinogen of interest, then the added dose D willhave a more substantial effect, R'. Thus, when a dose-response curve is concave upwards, orwhen there is a threshold dose below which no effect occurs, the presence of a backgroundexposure to carcinogens can increase the incremental risk associated with exposure to acarcinogen of interest.

As of today, we really do not have a very good idea of what levels of naturally occurringor cooking-induced carcinogens are in our food or how potent they are as carcinogens. Assuch knowledge continues to accumulate, it will be interesting to see what effect it has onsociety's attitudes and policies toward chemical carcinogens and mutagens in the environment.

References and Notes1. Ames, BN, McCann, J, and Yamasaki, E: "Methods for Detecting Carcinogens and Mutagens With the

Salmonella/Mammalian-Microsome Mutagenicity Test." Mutat Res 31, 347-364, 1975.2. Maron, D, and Ames, BN: "Revised Methods for the Salmonella Mutagenicity Assay." Mutat Res 113, 173-

215, 1983.3. McCann, JE, Choi, E, Yamasaki, E, and Ames, BN: "Detection of Carcinogens as Mutagens in the Salmonella/

Microsome Test: Assay of 300 Chemicals." Proc Natl Acad Sci USA 72, 5135-5139, 1975.4. McCann, JE, and Ames, BN: "Detection of Carcinogens as Mutagens in the S a l m o n e l l a / M i c r o s o m e Test:

Assay of 300 Chemicals: Discussion." Proc Natl Acad Sci USA 73, 950-954, 1976.5. Purchase, IFH, Longstaff, E, Ashby, J, Styles, JA, Anderson, D, Lefevre, PA, and Westwood, FR: "An

Evaluation of 6 Short-term Tests for Detecting Organic Chemical Carcinogens." Br J Cancer 37, 873-959,1978.

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Vi

OE3

73a373j=+•»

»mm»

O B'+D

DoseFigure 4. The low-dose portion of a hypothetical dose-response curve for a carcinogen showing the response (J?) toa dose CD) of a carcinogen and the greater response (R1) to the dose (D) when it is administered in the presence ofa background exposure (£') to carcinogens that work by the same mechanism as does dose (D).

6. Simmon, VF: "In Vitro Mutagenicity Assays of Chemical Carcinogens and Related Compounds With Salmonellatyphimurium." JNCI 62, 893-899, 1979.

7. Sugimura, T, Sato, S, Nagao, M, Yahagi, T, Matsushima, T, Seino, Y, Takeuchi, M, and Kawachi, T: "Over-lapping of Carcinogens and Mutagens." In Magee, PN, Takayama, S, Sugimura, T, and Matsushima, T (eds):Fundamentals in Cancer Prevention. Baltimore: University Park Press, 1976, pp. 191-215.

8. Seppilli, A, and Scassellati Sforzolini, G: "Sulla Presenza di Idrocarburi Policiclici Cancerigeni nelle CarniCotte alla Graticola." Boll Soc Ital Biol Sper 39, 110-114, 1963.

9. Lijinski, W, and Shubik, P: "Benzo(a)pyrene and Other Polynuclear Hydrocarbons in Charcoal-Broiled Meat."Science US, 53-55, 1964.

10. Lijinski, W, and Ross, AE: "Production of Carcinogenic Polynuclear Hydrocarbons in the Cooking of Food."Food Cosmet Toxicol 5, 343-347, 1967.

11. Nagao, M, Honda, M, Seino, Y, Yahagi, T, and Sugimura, T: "Mutagenicities of Smoke Condensates and theCharred Surface of Fish and Meat." Cancer Lett 2, 221-226, 1977.

12. Nagao, M, Honda, M, Seino, Y, Yahagi, T, Kawachi, T, and Sugimura, T: "Mutagenicities of Protein Pyro-lysates." Cancer Lett 2, 335-340, 1977.

13. Matsumoto, T, Yoshida, D, Mizusaki, S, and Okamoto, H: "Mutagenic Activity of Amino Acid Pyrolyzatesin Salmonella typhimurium TA98." Mutat Res 48, 279-286, 1977.

14. Nagao, M, Yahagi, T, Kawachi, T, Seino, Y, Honda, M, Matsukura, N, Sugimura, T, Wakabayashi, K, Tsuji,K, and Kosuge, T: "Mutagens in Foods, and Especially Pyrolysis Products of Protein." In Scott, D, Bridges,BA, and Sobels, FH (eds): Progress in Genetic Toxicology. New York: Elsevier/North-Holland, 1977, pp. 259-264.

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Containing Beef Extract." J Toxicol Environ Health 4, 189-202, 1978.18. Hargraves, WA, and Panza, MW: "Purification and Mass Spectral Characterization of Bacterial Mutagens

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Beef Extract and in Cooked Ground Beef. Use of Blue-Cotton for Efficient Extraction." Gann 74, 472-482,1983.

21. Felton, JS, Knize, MG, Wood, C, Wubbles, BJ, Healy, SK, Stuenner, DH, Bjeldanes, LF, Kimble, BJ, andHatch, FT: "Isolation and Characterization of New Mutagens From Fried Ground Beef." Carcinogenesis 5,95-102, 1984.

22. Weisburger, JH, Spingarn, NE, Wang, YY, and Vuolo, LL: "Assessment of the Role of Mutagens and En-dogenous Factors in Large Bowel Cancer." Cancer Bull 33, 124-129, 1983.

23. Pariza, MW, Loretz, LJ, Storkson, JM, and Holland, NC: "Mutagens and Modulator of Mutagenesis in FriedGround Beef." Cancer Res 43 (Suppl.), 2444s-2446s, 1983.

24. Kasai, H, Yamaizumi, Z, Wakabayashi, K, Nagao, M, Sugimura, T, Yokoyama, S, Miyazawa, T, Spingam,N, Weisburger, JH, and Nisimura, S: "Potent Novel Mutagens Produced by Broiling Fish Under NormalConditions." Proc Jpn Acad 56 (Series B), 278-283, 1980.

25. Kasai, H, Nisimura, S, Wakabayashi, K, Nagao, M, and Sugimura, T: "Chemical Synthesis of 2-Amino-3-methylimidazo[4,5-f]quinoline (IQ), a Potent Mutagen Isolated From Broiled Fish." Proc Jpn Acad 56 (SeriesB), 382-384, 1980.

26. Kasai, H, Yamaizumi, Z, Shiomi, T, Yokoyama, S, Miyazawa, T, Wakabayashi, K, Nagao, M, Sugimura, T,and Nishimura, S: "Structure of a Potent Mutagen Isolated From Fried Beef." Chem Lett, 485-488, 1981.

27. Sugimura, T: "Mutagens, Carcinogens, and Tumor Promoters in Our Daily Food." Cancer 49, 1970-1984,1982.

28. Rosenkranz, HS, and Mermelstein, R: "Mutagenicity and Genotoxicity of Nitroarenes: All Nitro-ContainingChemicals Were Not Created Equal." Mutat Res 114, 217-267, 1983.

29. Hosaka, S, Matsushima, T, Hirono, I, and Sugimura, T: "Carcinogenic Activity of 3-Amino-l-methyl-5.ff-pyrido(4,3-6]indole (Trp-P-2), a Pyrolysis Product of Tryptophan." Cancer Lett 13, 23-28, 1981.

30. Takayama, S, Nakatsuru, Y, Masuda, M, Ohgaki, H, Sato, S, and Sugimura, T: "Demonstration of Carcino-genicity in F344 Rats of 2-Amino-3-methylimidazo[4,5-/]quinoline From Broiled Sardine, Fried Beef and BeefExtract." Gann 75, 467-470, 1984.

31. Matsukura, N, Kawachi, T, Morino, K, Ohagaki, H, Sugimura, T, and Takayama, S: "Carcinogenicity in Miceof Mutagenic Compounds From a Tryptophan Pyrolyzate." Science 213, 346-347, 1981.

32. Takayama, S, Masuda, M, Mogami, M, Ohgaki, H, Sato, S, and Sugimura, T: "Induction of Cancers in theIntestine, Liver, and Various Other Organs of Rats by Feeding Mutagens From Glutamic Acid Pyrolysate."Gann 75, 207-213, 1984.

33. Ohgaki, H, Matsukura, N, Morino, K, Kawachi, T, Sugimura, T, and Takayama, S: "Carcinogenicity in Miceof Mutagenic Compounds From Glutamic Acid and Soybean Globulin Pyrolysates." Carcinogenesis 5, 815-819, 1984.

34. Yamaguchi, K, Shudo, K, Okamoto, T, Sugimura, T, and Kosuge, T: "Presence of 3-Amino-1,4-dimethyl-5H-pyrido[4,3-6]indole in Broiled Beef." Gann 71, 745-746, 1980.

35. Abrams, IJ: "Specialized Data File Reports on Food Intake—Menu Census 1977-1978." Unpublished dataobtained under FDA contract No. 223-79-2243 from MRCA Information Services, Northbrook, IL, February1984.

36. Yamaizumi, Z, Shiomi, T, Kasai, H, Nishimura, S, Takahashi, Y, Nagao, M, and Sugimura, T: "Detection ofPotent Mutagens, Trp-P-1 and Trp-P-2, in Broiled Fish." Cancer Lett 9, 75-83, 1980.

37. Elliger, CA, Henika, PR, and MacGregor, JT: "Mutagenicity of Flavones, Chromones and Acetophenones inSalmonella typhimurium: New Structure-Activity Relationships." Mutat Res 135, 77-86, 1984.

38. Nagao, M, Morita, N, Yahati, T, Shimizu, M, Masanori, K, Fukuoka, M, Yoshihira, K, Natori, S, Fujino, T,and Sugimura, T: "Mutagenicities of 61 Flavonoids and 11 Related Compounds." Environ Mutagen 3, 401-419, 1981.

39. Brown, JP, and Dietrich, PS: "Mutagenicity of Plant Flavonols in the Salmonella/Mammalian MicrosomeTest. Activation of Flavonol Glycosides by Mixed Glycosidases From Rat Cecal Bacteria and Other Sources."Mutat Res 66, 223-240, 1979.

40. Bjeldanes, LF, and Chang, GW: "Mutagenic Activity of Quercetin and Related Compounds." Science 197,577-578, 1977.

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43. Tamura, G, Gold, C, Ferro-Luzzi, A, and Ames, BN: "Fecalase: A Model for Activation of Dietary Glycosides

to Mutagens by Intestinal Flora." Proc Natl Acad Sci USA 77, 4961-4965, 1980.

44. Maruta, A, Knaka, K, and Umeda, M: "Mutagenicity of Quercetin and Kaempferol on Cultured Mammalian

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45. Van der Hoeven, JCM, Bruggeman, IM, and Debets, FMH: "Genotoxicity of Quercetin in Cultured Mammalian

Cells." Mutat Res 136, 9-21, 1984.

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