Caballero B. (ed.) Encyclopaedia of Food Science, Food Technology and Nutrition. Ten-Volume Set

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CARCINOGENS

Contents

Carcinogenic Substances in Food: Mechanisms

Carcinogenicity Tests

Carcinogenic Substances in

Food: Mechanisms

R Mehta and T J Schrader, Sir Frederick Banting

Research Centre, Ottawa, Ontario, Canada

Background

0001 Cancer is a major disease resulting in more than 7

million deaths per year worldwide. The risk of cancer

varies from region to region, suggesting a role for

geographic, environmental, and cultural factors in

cancer causation. Current figures suggest that

although about 37% of cancers deaths in the USA

are caused by cigarette smoking, diet is also a signifi-

cant risk factor, estimated to be associated with about

35% of cancer deaths.

0002 The human diet, whether derived from an animal

or a plant source, is an extremely complex and a

variable mixture of macro- and micronutrients, and

naturally occurring and synthetic substances. Studies

in experimental animals and human epidemiology

have shown that cancer development is a multistep

process consisting of three main stages: initiation,

promotion, and progression (Figure 1). Each stage

further involves numerous events, many of which

have as yet not been defined precisely. Generally,

during the initiation stage, normal cells are exposed

to chemical, viral, or physical carcinogens. Such

exposure may cause change(s) in the genome, provid-

ing a selective growth advantage to the altered

cells. Promotion involves selective growth of these

genetically altered cells leading to precancerous

lesions and eventually tumors. In the progression

stage, the tumor undergoes additional genetic and

morphological changes, making it malignant with

the ability to invade and spread (or metastasize) to

other sites in the body. Natural or synthetic carcino-

gens in food may contribute to cancer development

during either the initiation or promotion stage,

depending on the type of reactivity with the genetic

material.

Naturally Occurring Carcinogens in Food

0003Environmental exposures to naturally occurring

chemicals result principally from the food we

consume (1.5 kg day

1

). Although air and water

frequently contain at least trace levels of man-made

contaminants, they are seldom a source of naturally

occurring carcinogens. The numbers and amounts

of naturally occurring substances in a food far

exceed the synthetic compounds, and include

macronutrients (fat, carbohydrates, and proteins),

micronutrients (vitamins and minerals), and non-

nutrient constituents.

0004Both macro- and micronutrients have been shown to

modify cancer incidence in experimental animals. In

man, the modulation of cancer by macro- and micro-

nutrients is less definitive, though there is some evi-

dence that breast cancer rates are associated with the

amount and type of fat as well as with a high propor-

tion of calories as fat. In experimental models, total

caloric intake, as opposed to a single macronutrient, is

also known to have an impact on age-related changes in

tumor incidence. For example, decreasing calorie

intake results in a reduction in cancer incidence.

0005The naturally occurring chemical nonnutrients,

also termed natural food contaminants, may originate

in the food supply as a result of (1) physiological and

biochemical processes inherent in the food organisms

themselves; (2) byproducts formed during stress, stor-

age, processing, and preparation of food; (3) infection

or spoilage during storage by bacteria or fungi; (4)

materials introduced into the food product through

the dietary source consumed by the animal destined

for food; and (5) naturally occurring substances isol-

ated from raw plants or animal sources and intention-

ally added back to the food, for example, as a flavor or

color enhancer. Examples of carcinogens naturally

occurring in normal human diet under each of these

categories are shown in Table 1.

Synthetic Carcinogens in Food

0006Synthetic chemicals in foods occur as (1) direct or

intentional additives where synthetic chemicals are

added deliberately to enhance the appearance, nutri-

906 CARCINOGENS/Carcinogenic Substances in Food: Mechanisms

tional quality, or shelf-life of a food product by, for

example, adding color, a nutritional supplement,

or a preservative; and (2) indirect additives where

synthetic chemicals are present as a result of the use

of, for example, pesticides, packaging materials, anti-

biotics, or growth-promoting substances during food

production, processing, or storage. Indirect additives

may also be termed synthetic food contaminants.

Table 2 lists examples of carcinogens under these

categories.

Exposure and Risk Evaluation

0007The identification, monitoring, and categorization of

actual carcinogenic risks in relation to the types and

amounts of natural and synthetic substances in the

tbl0001 Table 1 Naturally occurring substances that may be present in US diets

Originof natural substancein food Examples (partiallist)

(1) Physiologically/biochemically produced

through processes inherent in the food

organism

Acetaldehyde, caffeic acid, estrone, estradiol, projestrone,

safrole, styrene, testosterone, 8-methoxypsoralen (with UV light

exposure)

(2) Byproducts formed during stress, storage,

food processing, and preparation.

Polycyclic hydrocarbons (benzo[a]pyrene), nitrosoamines

(N-nitrosodimethylamine), heterocyclic aromatic amines

(PhIP, MeIQx, IQ, glu-P-1), methylmercury compounds

(3) Infection or spoilage during storage by

bacteria or fungi

Aflatoxins, ochratoxin A, fumonisins, sterigmatocystin

(4) Introduction into food through dietary

source consumed by the organism destined

for food

Arsenic, cadmium, benz[a]anthracene, benzo[a]pyrene,

seafood toxins, indeno(1,2,3)pyrene, toxol in snakeroot,

aflatoxin from cows’ milk

(5) Natural substances isolated from raw plants or animal

sources and intentionally added back to the food

Sucrose, glucose, isolated soy protein flavors extracted or

distilled from spices, gums, corn, or tapioca starches

Initiation Promotion

Chemical

Deactivation

Excretion

Activation

Radiation

Viruses

Normal

cell

Initiated

cell

Preneoplastic

lesion

Malignant

tumour

Genetic

change

Cell

heterogeneity

Metastases

DNA, RNA,

Protein

Genetic

change

Apoptosis,

Necrosis

Selective clonal

expansion

Reactive

oxygen

Dead

cell

Progression

Increased cell proliferation

Decreased apoptosis

Resistance to cytotoxicity

Defects in growth control and cell cycle

Altered gap junctional intracelluar communication

Defects in terminal differentiation

Activation of protooncogenes

Inactivation of tumor suppressor genes

Receptor activation (e.g. estrogen, PPAR)

Lipid peroxidation and oxidative DNA damage

Disruption of signal transduction

Altered immune function

fig0001 Figure 1 General scheme for sequential steps in multistage carcinogenesis.

CARCINOGENS/Carcinogenic Substances in Food: Mechanisms 907

food are complicated by (1) the large variety of such

materials that are present in the human diet and (2)

the ability of such substances to manifest both

carcinogenic and anticarcinogenic properties with

different mechanisms of action.

0008 When comparing possible carcinogenic risks of

natural relative to synthetic substances, it is therefore

necessary to take into account (1) their relative expos-

ure through food and (2) any theoretical differences

in chemical and biological properties of both types of

chemicals. Traditional foods (e.g., grains, fruits, vege-

tables, and meat) comprise the bulk of the diet. Items

such as sugar and salt, generally recognized as safe,

are the most highly used direct food additives. Other

direct additivies such as artificial sweeteners, colors,

preservatives, spices, and flavors are used in much

smaller amounts. In contrast, indirect food additives,

such as pesticides and substances from packaging

materials, represent over 2000 chemicals, many of

which may be present in food below the level of

detection.

0009 In terms of possible differences in chemical prop-

erties, one commercially desirable property for a

synthetic food substance is chemical stability, which

is often achieved by adding a chlorine group to

the synthetic chemical. This may then give rise to

persistence of such chemicals in the environment,

and accumulation in the plant and animal food

chains.

0010 An excellent example of such persistent indirect

food additives is pesticides, which, among the general

public and regulatory agencies, have raised the most

concern compared to other indirect food additives.

Pesticides in food may originate from any of the

following sources: agricultural residues persisting on

the foodstuffs or in the environment, chemicals used

during storage, or water used in food preparation.

While animal studies provide evidence for the car-

cinogenicity of many pesticides, most of the data for

an association between pesticides and human cancer

have been obtained from occupational exposure stud-

ies in farmers. The findings of residues of pesticides

and their metabolites in blood, urine, breast milk, and

adipose tissues of individuals in the general popula-

tion, however, have sparked concerns over health

effects of pesticides. Certain subpopulations, in par-

ticular, may be susceptible to higher than normal rates

of exposure through the food supply. For example,

children are one vulnerable subpopulation that may

be exposed to significantly greater amounts of pesti-

cide residues through placental transfer and breast

milk. Another subpopulation of current concern is

that of the Arctic whose marine food chain has accu-

mulated persistent residues of organochlorine pesti-

cides that were heavily used in southern parts of the

North American continent until the 1980s. Such mi-

gration of persistent, indirect food additives across

continents may result in adverse health consequences

for the Arctic indigenous populations that consume

marine mammals as part of their traditional diet.

0011An example of a group of naturally occurring car-

cinogens in food that have been most studied and

regulated include the mycotoxins such as aflatoxins,

ochratoxins, and fumonisins. Aflatoxins, produced

primarily by strains of Aspergillus flavus, have been

found as fungal contaminants in peanuts, corn,

wheat, rice, and foods made from these commodities.

An association between dietary aflatoxin exposure

and high incidence of liver cancer has been observed

in a number of African and South-east Asian coun-

tries. Of all the aflatoxins, aflatoxin B

is the most

potent liver carcinogen. Recent studies have shown

that other factors such as hepatitis B viral (HBV)

infection, alcohol consumption, smoking, and other

tbl0002 Table 2 Synthetic substances that may be present in US diets

Origin of synthetic substance in food Examples (partiallist)

(1) Directly or intentionally

added

Appearance modifiers: Glazes, waxes, colors (tartrazine, sunset yellow), surface finishing

agents (polyvinyl-pyrrolidone)

Curing and pickling agents: Sodium nitrite, ascorbic acid, sodium tripolyphosphate

Nutrient replacements/supplements: Sucrose polyesters, all essential vitamins, minerals

and fatty acids

Sweeteners: Saccharin, aspartame, sucrose, glucose

Product stability aids: Antioxidants (butylated hydroxyanisole), antimicrobials (sodium

benzoate, potassium sorbate)

Flavoring agents: Triethyl citrate, monosodium glutamate, ethyl maltol

(2) Indirectly or unintentionally

added

Pesticides: Toxaphene, chlordane, organotins, triazines, dinitranilines, arsenicals and

fluorides, DDT, chlordecone

Growth promoters: Estradiol, progesterone, reserpine, testosterone

Antibiotics: Sodium o-phenyphenate

Packaging materials: Acrylamide, di-2-ethylhexylphthalate, carbon tetrachloride, chloroform,

styrene, styrene oxide, dimethylformamide

908 CARCINOGENS/Carcinogenic Substances in Food: Mechanisms

environmental variables also play a role in liver

cancer induction by aflatoxin B

. In Western coun-

tries, human exposure to aflatoxin B

is mainly from

nuts and nut products, and for children from peanut

butter, estimated to be 1–2 ng per kilogram body

weight (bw) per day according to Canadian estimates

(1985–1987). This intake level is about 10-fold

higher than the Canadian set tolerable daily intake

(TDI) of 0.11–0.19 ng per kilogram bw per day. This

TDI was based on a carcinogenic risk level of

1:100 000, as estimated from epidemiological data

obtained from Asian and African populations. How-

ever, such an exposure level is not considered a health

risk for Canadians because HBV infection, another

associated factor in liver cancer development by afla-

toxin B

, is not endemic in Canada, unlike the Asian

and African countries.

0012 Another mycotoxin, ochratoxin A, produced by

Aspergillus ochraceus and related species, has been

found to occur in foods of plant origin such as wheat,

barley, oats, corn, dry beans, and animal-derived food

products. It has also been detected in human blood,

milk, and tissues from European and Canadian popu-

lations, suggesting widespread human exposure.

However, various Western countries have regulations

to ensure that levels in food are controlled at levels

below the estimated TDI of 1.5–5.7 ng per kilogram

bw per day, which is based on a National Toxicology

Program carcinogenicity study. Ochratoxin A has

been implicated in endemic nephropathies in Euro-

pean populations as well as livestock, and has been

shown to cause renal cancer in two rodent species.

0013 Fumonisins, produced by Fusarium moniliforme

and F. proliferatum, are fungal contaminants primar-

ily found in corn and corn-based food products. Six

fumonisins have been isolated and characterized

from F. moniliforme, and are designated as fumonisin

, and A

. Only fumonisin B

and B

have been studied to a significant extent. The con-

sumption of corn contaminated with fumonisins has

been associated with a high incidence of esophageal

cancer in human populations in the Transkei region

of South Africa and Henan Province of Northern

China. Fumonisin B

has also been shown to cause

liver cancer in rats and female mice, and renal tubule

neoplasms in rats. Levels of fumonisin B

vary from

year to year, based on fungal growth conditions (e.g.,

moisture during the growing season), but are consist-

ently in the 0.5–2 p.p.m. range in US cornmeal and

have been reported to be as high as 150 p.p.m. in corn

for human consumption in South Africa. The levels of

this mycotoxin in some foods also change as a result

of the cooking process through hydrolysis of the tri-

carballylic acid groups, or by reacting with reducing

sugars producing (carboxymethyl)-fumonisin B

Overall, it has been difficult to determine the accurate

average daily consumption of fumonisin B

in the

USA because this will depend on the level of contam-

ination, the diet portion that is corn or corn-based

foods, and the cooking habits. In terms of risk man-

agement, the TDI for these group of mycotoxins has

not been established owing to a lack of sufficient

toxicity data, although Switzerland has a regulatory

limit of 1 p.p.m. in human food.

0014Examples of naturally occurring carcinogens that

may originate in the food supply as byproducts

during food processing and preparation include

the nitrosamines, polycyclic aromatic hydrocarbons

(PAHs), heterocyclic amines (HCAs), and lipid oxida-

tion products. These compounds are present in some

cooked foods, particularly fried, grilled, or barbecued

meat and fish products. Exposure to these products

can also arise from food processing: curing, drying,

smoking, roasting, refining, and fermentation. The

most commonly detected PAHs are benzo[a]pyrene,

benzo[a]anthracene and dibenzo[a,h]anthracene, of

which benzo[a]pyrene is the most carcinogenic. The

intake of PAHs in Europe, the USA, and Japan has

been estimated to be around 1 mg per person per year,

and for benzo[a]pyrene, the values range from 0.01

to 0.61 mg per person per year. Of this, only about

25–30% of the PAHs originates from food, with

the majority coming from air pollution and smoking.

PAHs have been considered probable human carcino-

gens based on animal and in-vitro mutagenicity data.

There is no definitive evidence for a possible relation-

ship between ingestion of PAH-contaminated food

and human cancer.

0015The main forms of N-nitroso compounds in food

are nonvolatile, including a large number of com-

pounds that could be potentially formed, e.g., pro-

teins containing N-nitrosated peptide linkages, such

as N-nitrosoproline. Although nonvolatile N-nitroso

compounds have not been shown to be carcinogenic

or mutagenic, they could be precursors to the volatile

carcinogenic nitrosamines. For example, during

food processing, preparation, or preservation, added

nitrite and/or nitrate, naturally occurring nitrite, or

nitrogen oxides resulting from heating or drying

of foods with combustion gases can react with

amino compounds to produce N-nitroso compounds.

Examples of the volatile carcinogenic nitrosamines

that occur in food include N-nitrosodimethylamine,

(NDMA), N-nitrosodiethylamine, N-nitrosopyrroli-

dine, and N-nitrosopiperidine. The evidence that

some N-nitroso compound are carcinogenic is over-

whelming from animal studies. In epidemiological

studies, positive correlations have been observed

between the high intake of nitrosamines, and inci-

dences of esophageal and nasal cavity cancers in

CARCINOGENS/Carcinogenic Substances in Food: Mechanisms 909

China, stomach cancer in Japan and Norway, and

colorectal cancer in Finland.

0016 A variety of HCAs are produced as pyrolysis

products of sugars, amino acids, creatinine, and

other constituents when fish and meat products

are subjected to high temperatures during normal

cooking processes. The extent of HCA exposure

(estimated at nanogram to microgram levels per day

per person) is influenced by factors such as type of

fish or meat ingested, frequency of consumption,

portion size, method of cooking, cooking tempera-

ture and duration, and the amounts of the pan

residues and gravy ingested. The different groups of

HCAs identified include pyrido-imidazoles or indoles

(Trp-P-1, Trp-P-2, AaC, MeAaC); derivatives of

imidazoquinolines or IQ compounds (IQ, MeIQ);

imidazoquinoxalines (Iqx, MeIQx, DiMeIQx); and

the imidazopyridines (PhIP). Among the various

HCA contained in cooked meat, the content of PhIP

is highest, followed by MeIQx in the Western diet. In

urine samples from Japanese volunteers, however, the

levels of MeIQx are higher than those of PhIP. Long-

term animal studies have shown multisite carcino-

genic effects with major target organs being the

liver, colon, and mammary gland in mice and rats

for MeIQx and PhIP, respectively, and liver for IQ in

cynomolgus monkeys. Epidemiological studies have

focused on meat consumption and several types of

human cancer, but a major challenge has been to

differentiate the effects of HCAs from that of meat

by itself and other confounding factors. DNA adducts

in human tissues have also been detected.

0017 Lipid oxidation products, particularly cholesterol

oxide products, are common in foods exposed to

severe cooking conditions such as deep-fat frying

and pan frying following long-term storage in a

freezer or in dehydrated form. The formation of chol-

esterol oxide products is greatly accelerated by auto-

oxidation of coexisting unsaturated triacyglycerols,

resulting in oxidized forms or epoxides of cholesterol.

Several studies, for example, have reported high levels

(6.8–58.8 p.p.m.) of cholesterol oxide products in

french fries cooked in animal fats or mixtures of

vegetable oils and animal fats. In terms of risk, the

relationship between long-term consumption of lipid

oxidation products and human health is not clear.

Data from animal studies are not reliable, because

high levels (10–15% of diet) of lipid oxidation

products are required in the diet of the laboratory

animal to produce an effect.

0018 Following a number of evaluations, the Inter-

national Agency for Research on Cancer has con-

cluded that several of the nitrosamines, PAHs, and

HCAs are probable or possible human carcinogens

based on both long-term animal studies and in vitro

mutagenicity tests. However, in the absence of suffi-

cient evidence that these mutagens really cause

human cancer, no limits have been set for their pres-

ence in foods. The regulatory agencies in most de-

veloped countries recommend minimizing their

occurrence in foods by changing cooking practices.

0019In the USA, UK, Canada, and many other coun-

tries, food-safety legislation is based on traditional

food, added substances, and unintended added sub-

stances. It would seem, therefore, that food additives

and pesticides do not pose a major cancer threat in

view of their rigorous regulation, and hence, only

limited exposure is expected from below ‘tolerance

level’ quantities being permitted in foods. In the

USA, the Food Additive Amendment in 1958 con-

tained the ‘Delaney Clause’, which states that no

natural of synthetic additive be allowed in food in

any amount if it has been shown to produce cancer

in animal studies or other appropriate tests. There is

no legislation, however, to regulate naturally occur-

ring carcinogens from plants or cooking practices, or

the macrocomponents of the diet which with im-

proved analytical techniques have been shown in

recent years to be a cancer risk, either directly or by

modulating the disease process. Attempts to estimate

cancer risks from naturally occurring carcinogens

are further complicated because of the difficulties in

differentiating between the effects of these substances

from those of other confounding factors such as indi-

vidual differences within human populations with

respect to rates of exposure, the ability to activate

and detoxify these substances, the presence of other

protective mechanisms, and lifestyle. An in-depth

understanding of the biological mechanisms by

which these diverse carcinogens exert their action is

therefore necessary for the development of methods

and biological markers that allow (1) reliable

extrapolation of biological mechanism-related data

from appropriate experimental models to humans;

(2) accurate biomonitoring in human populations

for exposure to specific food borne carcinogens and

their health effects; and (3) estimations of cancer

susceptibility to specific foodborne substances in in-

dividuals and subpopulations. Such measures would

then provide essential data for assessment of potential

human cancer risk as well as perhaps avenues for

legislative control of these substances.

Mechanisms of Carcinogenesis

0020Although the detailed molecular mechanisms of

cancer induction by carcinogens in food remain to

be discovered, several factors required for the multi-

step process have been identified: two or more genetic

events, either inherited or resulting from point

910 CARCINOGENS/Carcinogenic Substances in Food: Mechanisms

mutations, chromosomal rearrangements, insertions

or deletions of genes, and gene amplification; cell

replication to incorporate the altered DNA into the

genome; cell proliferation to selectively expand the

altered cell population into a benign tumor; and

additional genetic events leading to a malignant

tumor with the ability to invade and metastasize to

other sites in the body. Some of the genetic events at

various stages during cancer development include

overexpression and/or activation of cellular protoon-

cogenes, and deletion or inactivation of tumor sup-

pressor genes (Figure 1). Such alterations in the

expression of protooncogenes and tumor suppressor

genes may then cause the dysregulation of the cell

cycle either directly or indirectly. The cell cycle is a

complex process involved in the growth and prolifer-

ation of cells, and involves numerous regulatory pro-

teins that direct the cell through a specific sequence of

events concluding with mitosis and the production

of two progeny cells. Many of the protooncogenes

and tumor-suppressor genes also play a key role

in the regulation of natural cell death or apoptosis.

Over the past decade, it has become increasingly clear

that cancer is the disease not only of increased cell

replication but also of inhibition of apoptosis. The

relationship between cell replication and normal cell

death (or loss) determines the rate of tumor growth.

In addition, the immune system is known to play a

role in recognition of cancer cells and may contribute

to cancer prevention by killing cancer cells. There-

fore, any inhibition of the immune surveillance mech-

anism may compromise its protective capacity and

thus result in tumor development.

0021Natural or synthetic carcinogens in food can

generally be divided into those that directly affect

DNA (genotoxic) and those that do not (nongeno-

toxic), and may be classified according to their role

in triggering events during one or more of the stages

of cancer induction (Table 3). Genotoxic substances

chemically react with DNA and are usually impli-

cated during the initiation stage of carcinogenesis.

Nongenotoxic carcinogens, which do not have the

capacity to react with the DNA directly, may alter

DNA through indirect or epigenetic mechanisms and

are, therefore, expected to contribute during the

promotion or progression stages. Some chemicals

cause indirect DNA damage and genomic alteration

through oxygen radical generation in a variety of

ways. Certain nongenotoxic carcinogens, such as

peroxisome proliferators and estrogens, interact

with specific receptor proteins to produce protein–

ligand complexes, which then interact with, and

tbl0003 Table 3 Possible mechanisms of carcinogenicity by foodborne substances

Carcinogen classification

accordingto cancer stage of

most likely activity

Capacity to Possible mechanism(s) of action Selected examples

Initiating agent Initiate cells only Electrophile generation

through spontaneous

breakdown or metabolic

activation of chemical with

resultant DNA damage and

two or more genomic

mutations

Mycotoxins (aflatoxin B

)

Nitrosamines (NDMA)

Heterocyclic amines

(PhIP, IQx)

Pesticides (toxaphene)

Promoting agent Selectively expand the

initiated cell population

Convert an initiated cell or a

promoted cell to a

malignant cell

One or more of:

. enhanced cell proliferation

. decreased apoptosis

. epigenetic effects via:

. lipid peroxidation and

oxidative DNA damage

. receptor protein

interaction–peroxisome

proliferation, hormonal action

. altered signal transduction

pathways

. compromised immune function

Mycotoxins (fumonisin B

)

Pesticides (chlordane,

organochlorines,

chlorophenols)

Packaging materials

(di-(2-ethylhexyl)phthalate)

Complete carcinogen Induce cancer from normal

cells usually exhibiting

the properties of initiating,

and promoting agents

Electrophile generation,

DNA damage,

genomic mutations, and any or

all of the above tumor-promoting

mechanisms

Mycotoxins (aflatoxin B

fumonisin B

)

Nitrosamines

Heterocyclic amines

Pesticides

Some promoting agents may also exhibit weak initiating capacity with sufficient promoting ability to produce tumors, in which case, they may also be

classified as complete carcinogens, e.g., Fumonisin B

CARCINOGENS/Carcinogenic Substances in Food: Mechanisms 911

modify, the expression of critical regions of genomic

DNA. Other substances do not appear to exert any of

the above effects, yet greatly enhance cell prolifer-

ation, and are usually active during the promotion

stage (Table 3).

Metabolic Activation and Electrophile Generation

0022 Many of the genotoxic foodborne carcinogens de-

scribed above undergo metabolic activation and

transformation in the body. Through oxidative

metabolism primarily by the cytochrome P450 family

of mixed-function oxidase enzymes, highly reactive

electron-deficient species can be produced, which can

then depurinate or deaminate DNA, or form covalent

adducts with cellular macromolecules, including

DNA. Electrophiles may also be produced through

flavine-dependent enzymes, the prostaglandin endo-

peroxide synthetase system, and other peroxidases.

These electrophiles may be further metabolized by

reaction with sulfate, glutathione, or glucuronic acid

(phase II reactions), resulting in detoxification and

excretion. In some cases, phase II reactions may also

lead to carcinogenic metabolites. Therefore, the over-

all dose of the carcinogen that interacts with DNA

(biologically effective dose) is often dependent on the

presence of a number of competing metabolic path-

ways in any given organ or animal species. The

following examples illustrate the diversity in organ

specificity and mechanisms of metabolic activation

leading to DNA interaction among naturally occur-

ring carcinogens originating from fungal contamin-

ation and cooking processes.

0023 Mycotoxins Aflatoxin B

, a liver carcinogen, is

metabolized via the cytochrome P450-catalyzed

oxidation to various hydroxylated derivatives, and

two unstable, highly reactive epoxide isomers. The

epoxide, aflatoxin B

-8,9-oxide is considered to

be the active metabolite, which covalently binds

to guanine in the DNA to form the major adduct

8,9-dihydro-8-(N

-guanyl)-9-hydroxyaflatoxin B

(AFB

-N

-GUA) (Figure 2).

0024 This epoxide can also react with RNA, histones,

and albumin. The balance between the various pri-

mary steps, the relative rates of detoxification of the

hydroxylated metabolites via glutathione or glucuro-

nic acid conjugation, and the extent of formation

and repair of the DNA adducts determine the carcino-

genicity of aflatoxin B

in different species. The

AFB

-N

-GUA formed may lead to persistence of

repair-resistant adducts and depurination or error-

prone DNA repair, which then result in single-strand

breaks, base-pair substitution, or frameshift muta-

tions. Mispairing of the adduct induces both transver-

sion and transition mutations. Such interactions may

also result in the activation of oncogenes and/or

inactivation of tumor suppressor genes. Thus, recent

human and experimental studies have indicated a

high frequency of G !T transversions, and the

prevalence of the codon 249 mutation in the tumor

suppressor p53 gene in liver tumors obtained from

populations where aflatoxin B

is the causative agent.

0025Ochratoxin A, a renal carcinogen, contains an iso-

coumarin moiety linked by a peptide bond to phenyl-

alanine and can be hydroxylated via P450 or

degraded by peptidase, as indicated in Figure 3.

0026Recent studies have provided clear evidence of

renal carcinogenicity of ochratoxin A in two rodent

species, and ochratoxin A–DNA adducts have been

detected in kidney, liver, and spleen of mice exposed

to the mycotoxin. Ochratoxin A was initially found

not to be mutagenic in various microbial and

mammalian gene mutation assays, although weak

genotoxic activity was observed to mammalian cells.

While the mechanism of carcinogenesis is unknown,

one toxic mechanism is the inhibition of protein

synthesis by competition with phenylalanine in the

phenylalanyl-tRNA synthetase-catalyzed reaction,

and inhibition of other enzymes that use phenylalan-

ine as a substrate such as phenylalanine hydroxylase.

The effect of ochratoxin A on protein synthesis is

followed by an inhibition of RNA synthesis. Ochra-

toxin A has also been found to enhance lipid

peroxidation both in vitro and in vivo.

0027Nitrosamines Through mechanistic studies, it is evi-

dent that nitrosamines are the most potent initiating

carcinogens as well as complete carcinogens. This is

because nitrosamines, through metabolic activation,

have the capacity to cause point mutations leading

to protooncogene activation by alkylation and base

substitution of critical sites in DNA. As an example,

the metabolic activation of NDMA is illustrated

(Figure 4).

0028N-Nitrosodimethylamine is oxidized by the cyto-

chrome P450 monooxygenase enzymes to hydroxy-

methyl-methylnitrosamine, which is unstable and

further decomposes to formaldehyde and nitro-

somethylamine. The latter then gives rise to nitrogen-

separated ion pairs and the unstable intermediate

methyldiazoniumhydroxide. The proximate species

that alkylate sites in protein, RNA, and DNA are

produced either directly (methyl cabonium ion) or

after loss of water (diazomethane) from methyldiazo-

niumhydroxide. More than 15 different sites suscep-

tible to alkylation of bases in DNA have been

identified. Alkylation at different sites appears to

have variable probabilities of generating specific

genotoxic events. For example, the miscoding

-alkylguanines are likely the predominant sources

912 CARCINOGENS/Carcinogenic Substances in Food: Mechanisms

O-demethylation

Aflatoxin B

-8,9-oxide

P448

P450

Hydroxylation

AFM

AFQ

AFP

Aflatoxin B

ReductionAflatoxicol

OCH

Nucleic acid

adducts

8,9-dihydro-8-(N

-guanyl)-

9-hydroxyaflatoxin B

(major DNA adduct formed

in vivo and in vitro)

8,9-dihydro-8-(N

-formyl-

2,5,6-triamino-4-oxopyrimidin-N

-YL)

9-hydroxy aflatoxin B

Epoxide

hydrase

(?)

8,9-dihydro-8,9-dihydroxy-

aflatoxin B

Glutathione

s-transferase

Aflatoxin-

glutathione

conjugate

OCH

DNA

OCH

fig0002 Figure 2 Metabolic activation of aflatoxin B

CARCINOGENS/Carcinogenic Substances in Food: Mechanisms 913

for mutations leading to activated oncogenes.

However, other adducts may also contribute at a

lower frequency or may enhance the effects of the

-alkylguanine adducts through DNA repair pro-

cesses that lead to incorporation of incorrect bases.

0029 Heterocyclic amines Studies in rodents and non-

human primates indicate that HCAs are rapidly

absorbed, widely distributed across tissues, exten-

sively metabolized and excreted via urine or feces.

The principal sites of HCA metabolism are shown

in Figure 5 with MeIQx as an example. These

compounds can be metabolized by cytochrome

P450-mediated ring oxidation or direct phase II con-

jugation reactions leading to detoxification. The cyto-

chrome P450-mediated N-oxidation of the exocyclic

amine nitrogen produces reactive metabolites that

bind to DNA, RNA, and protein in almost all tissues

studied. Figure 6 illustrates the structures of MeIQx

adducts with guanine in the DNA. Further activation

of the N-hydroxy HCA metabolites may occur by

phase II enzymes to reactive ester derivatives, which

are not stable and spontaneously rearrange into elec-

trophilic arylnitrenium ion intermediates that react

with DNA, RNA, or protein, and are mutagenic in

in vitro assays.

Phenylalanine

Ochratoxin α

Peptidase

Ochratoxin A

Cytochrome P-450

COOH

(4R)-4-hydroxyochratoxin A

(4S)-4-hydroxyochratoxin A

COOH

fig0003 Figure 3 Ochratoxin A and its metabolism.

914 CARCINOGENS/Carcinogenic Substances in Food: Mechanisms

0030 The mutagenicity of HCAs has also been attributed

to the DNA adducts formed from the arylnitrenium

ion intermediates. Interspecies, interindividual, and

tissue-specific differences in the expression of various

enymatic pathways influence DNA adduct formation

by HCAs. The levels of DNA adduct formation do

not always correlate with target organ specificity,

suggesting that, as with other genotoxic carcinogens,

other factors are necessary for tumor induction by

HCAs. These substances generally enhance cell pro-

liferation in their target organs: for example, PhIP,

a colon carcinogen in colon, but not liver or kidney;

and MeIQx, a hepatocarcinogen, in liver but not

colon or kidney. Inhibition of apoptosis, and

(CH

)

Carbonium

ion

Tight ion

pair

Diazomethane

DNA, RNA, Protein

Methyl-

diazonium ion

−

Syn-methyldiazo

hydroxide

−OH

−

P450

DMN

+ −

HOCH

fig0004 Figure 4 In vivo conversion of dimethylnitrosamine.

-glucuronidation

-hydroxylation

-sulfamation

-glucuronidation

-oxidation

-hydroxylation

O-sulfonation

O-glucuronidation

fig0005 Figure 5 Sites of heterocyclic amine metabolism (MeIQx is shown as an example) by cytochrome P450 and phase II enzymes. The

N-hydroxy HCA derivatives may undergo further activation by phase II enzymes.

CARCINOGENS/Carcinogenic Substances in Food: Mechanisms 915