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This chapter discusses the genotoxic elements in the human food chain in regard to cancers of the stomach, colon, and breast. Broiled and fried foods may be sources of genotoxic carcinogens associated with many of the nutritionally related cancers. Carcinogens designated as genotoxic are mutagens in the Ames bacterial indicator system and induce DNA repair in the Williams liver cell test system. Many kinds of vegetables contain appreciable amounts of certain mutagenic, polyhydroxylated flavones, such as quercetin or kaempferol. Metabolism of the mutagens found in fried meat is beginning to be studied by in vitro and animal experiments. Extensive Maillard reactions may yield mutagens and carcinogens. J. Sander found that such carcinogens were formed through the reaction of nitrite and suitably substituted secondary amines or amides. The fact that the formation of carcinogens may be blocked by vitamin C or by vitamin E is most important.

The majority of human cancers have multifactorial environmental causes stemming mainly from lifestyle factors such as use of tobacco products through cigarette smoking, snuff dipping, or chewing, and specific nutritional elements and dietary practices. The mechanisms of these lifestyle factors can be analyzed in terms of specific genotoxic carcinogens, and of epigenetic agents or promoting factors. Tobacco and tobacco smoke contain not only genotoxic carcinogens but also, with a more important ultimate effect, cocarcinogens and promoters. Alcohol acts as a cocarcinogen with tobacco, possibly by modifying the metabolism of carcinogens in select organs. Genotoxic carcinogens as nutritional factors may be found in pickled, salted, and smoked foods and may be responsible for gastric cancer. Vitamins C and E and other antioxidants are effective inhibitors. Other types of genotoxic carcinogens are mutagenic chemicals found in broiled and fried foods, and these may be involved in cancer of the colon, breast, and prostate. Promoting effects derive from a high level of dietary fat, which has been linked epidemiologically and through laboratory studies to a higher risk for these cancers. Possible mechanisms by which fat exerts its effects are an increased concentration of bile acids in the stool, as related to colon cancer, and which may be countered by a high cereal fiber diet, to increase stool bulk. In relation to breast or prostate cancer, fat may exert its effect on complex hormonal balances, and also on membrane composition. These promoting effects, whether associated with tobacco smoke or nutrition, are highly dose-dependent, and provided the insult is not too far advanced, reversible. Thus, lowering the dosage, or eliminating the effect as in smoking cessation should have an appreciable effect in reducing overt disease development, and do so fairly promptly. This may apply also to a reduction of second disease in cases where a first occurrence has been successfully treated by conventional means.

Geographic pathology provides information that a disease may have a quite different incidence and mortality as function of area of residence. Research in animals can model fairly precisely what is learned through geographic pathology, and provides the basis for understanding possible mechanisms of action. These approaches can yield public health recommendations and health promotion activities. Frequent intake of foods with saturated fats such as meat and dairy products raises the risk of coronary heart disease, especially so in smokers. The total mixed fat intake accounts for the high incidence of the nutritionally linked cancers, i.e. of the post-menopausal breast, distal colon, prostate, pancreas, ovary and endometrium, in Western countries, and of increasing impact in Asia, as people there adopt Western eating habits. Monounsaturated oils such as olive oil are low risk fats, as found in animal models, and through the fact that the incidence of coronary heart and neoplastic diseases is lower in the Mediterranean region, where such oils are customarily used. Fish and fish oils are protective, as is noted in countries with a high intake. The genotoxic carcinogens for a number of these cancers are heterocyclic amines that are produced during the broiling or frying of meaty foods that contain creatinine. Excessive salt intake can cause high blood pressure and with stomach cancer, especially with inadequate intake of potassium, from fruits and vegetables, and calcium from certain vegetables and low fat dairy products. Bran cereal fiber intake, especially with adequate calcium, yields an increased stool bulk, eliminating factors involved in colon and breast cancer. Vegetables and fruits, as well as soy products, are rich in antioxidants that are essential to lower disease risk stemming from reactive oxygen species in the body. Green and black tea are excellent sources of such beneficial antioxidants of a polyphenol nature, as is cocoa and chocolates. Antioxidants also extend healthy aging and may protect against Alzheimer's and Parkinson's diseases. Nutritional lifestyles can be described for most populations in the world that offer the possibility of a healthy long life.

Biomedical research has uncovered the mechanisms whereby tea promotes good health and lowers the risk of major chronic diseases, such as heart disease and many types of cancer. The active components in tea are polyphenols, epigallocatechin gallate in green tea, theaflavins and thearubigins in black tea. Green and black tea and the polyphenols have similar beneficial effects. The mechanisms are categorized into 5 groups. 1) Tea polyphenols are powerful antioxidants. They decrease the oxidation of LDL cholesterol and lower the risk of heart disease, and also inhibit action of reactive oxygen species mediating the oxidation of DNA associated with carcinogenesis 2) Tea polyphenols induce detoxifying enzymes, glucuronosyl transferases, eliminating active forms of carcinogens and other toxicants, accounting for the lower cancer risk. 3) Tea polyphenols lower duplication rates of cancer cells and inhibit the growth of cancer, increase apoptosis and lower angiogenesis. 4) Tea polyphenols alter the intestinal bacterial flora, suppressing undesirable bacteria and favoring growth of beneficial bacteria. 5) Aging phenomena, and diseases associated with the formation of reactive oxygen species (ROS) are inhibited.

The carcinogenic risk of aromatic amines in humans was first discovered when a physician related the occurrence of urinary bladder cancer to the occupation of his patients. They were employed in the dyestuff industry, chronically exposed to large amounts of intermediate arylamines. Laboratory investigations disclosed that rats and mice administered specific azo dyes arylamines or derivatives developed cancer, primarily in the liver. Also, at that time, a possible pesticide, 2-aminofluorene, was tested for chronic toxicity, revealing that it rapidly induced cancers in several organs of rodents. This led to investigations on the mode of action of this class of chemicals, including their metabolic conversion. Biochemical activation to more reactive N-hydroxy compounds was found to occur, mostly in the liver, through what is now known as the cytochrome p450 enzyme systems, and also through prostaglandin synthetases. There were species differences. Guinea pigs were resistant to carcinogenesis because of the low titer of the necessary activating enzymes. In target tissues, a second essential reaction was necessary, namely acylation or sulfate ester formation. The reactive compounds produced display attributes of genotoxicity in appropriate test systems. Interest in this class of compounds increased when of Sugimura and colleagues discovered the formation of mutagens at the surface of cooked meat or fish, that were identified as heterocyclic amines (HCAs). These compounds undergo the same type of activation reactions, as do other arylamines. Epidemiological data suggest that meat eaters may have a higher risk of breast and colon cancer. HCAs induced cancer in rats in these organs and also in the prostate and the pancreas. In addition, there is some evidence that they affect the vascular system. The formation of HCAs during cooking can be decreased by natural and synthetic antioxidants, by tryptophan or proline, or by removing the essential creatine through brief microwave cooking prior to frying or broiling. The amounts of HCAs in cooked foods are small, but other components in diet such as omega-6-polyunsaturated oils have powerful promoting effects in target organs of HCAs. On the other hand, the action of HCAs may be decreased by foods containing antioxidants, such as vegetables, soy, and tea. Some constituents in foods also induce phase II enzymes that detoxify reactive HCA metabolites. Additional mechanisms involved decreased growth of neoplasms by intake of protective foods. Possibly, the carcinogenic effect of HCAs is accompanied by the presence of reactive oxygen species (ROS), which are also inhibited by antioxidants. World-wide, there have been many contributors to knowledge in this field. Adequate information may permit now to adjust lifestyle and lower the risk of human disease stemming from this entire class of aryl and HCA.

Dunning began inbreeding, what is now the Fischer F344 rat, in 1931. Because of her publications showing a low incidence of spontaneous tumors to 35 months of age, we selected the F344 rat for most of the studies in the National Cancer Institute (NCI) Bioassay Program, beginning in 1964. We were surprised by the finding that untreated male F344 rats displayed a high incidence of Leydig cell tumors of the testes beginning at about 17 months of age. The key difference between the results of Dunning and the NCI Bioassay Program was that her animals were retired breeders, whereas the NCI studies utilized virgin rats. The question of breeding was, therefore, examined as a possible protective factor. Groups of male F344 CDF/Crl rats were kept as virgin animals, or permitted access to bilaterally tube ligated female F344/Crl rats that were replaced twice over a 52-week period. At that time, all males from both groups were housed three per cage to 85 weeks when they were killed in a CO 2 atmosphere and necropsied. Sections were prepared from the fixed tissues, stained and studied by histopathology. The results were evaluated by appropriate statistical methods. Virgin and sexually active F344 rats displayed monolateral or bilateral Leydig cell tumors. There was no statistical difference between the two groups. Despite the early difference between breeding and virgin F344 rats, a control experiment failed to disclose an effect of sexual activity on the occurrence of Leydig cell cancers. This disease displays some difference in incidence in various parts of the world, with the higher socioeconomic groups having a greater incidence. Etiological factors on the occurrence of this disease in animals and in humans remain to be discovered.

Observations on cancer causation are some 150 years old, but actual detailed research on elements bearing on cancer started at the beginning of the twentieth century. Rapid progress, however, is only some 40 years old. Studies in humans documented certain lifestyle related factors to lead to cancer, and research in animal models strengthened this information. With the realization that there are carcinogens that in a metabolically activated attack DNA, in contrast to other agents that act by promoting, enhancing processes through totally distinct mechanisms, it became possible to develop and apply tests for DNA reactivity, in a prokaryotic organism, the widely used Salmonella typhimurium test by Ames and in a eukaryotic system, namely freshly explanted liver cells displaying evidence of DNA repair by Williams. A battery of these two tests are over 90% accurate in defining genotoxicity. Virtually all documented human carcinogens are genotoxic. With advances in molecular biology, mutational events are traced to changes in tumor suppressor genes or in oncogenes, that can serve as markers of risk. In addition, reactive oxygen systems (ROS) are involved in both the early steps in cancer and in the developmental aspects. Thus, foods containing antioxidants such as vegetables, fruits, soy products, cocoa and tea that counteract ROS are protective in cancer causation and development. Worldwide application of current knowledge and mechanisms to cancer prevention, the definitive means of cancer control, is likely to lower not only cancer but also heart disease risk in the current century.

The effects of green tea on the metabolism of the food report that phenobarbital increased urinary glucuronides of carcinogen 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) hydroxylated metabolites of the carcinogen 2-acetylaminowith emphasis on the formation of the detoxified glucuron- fluorene (N-2-fluorenylacetamide), accounting for its decreased ides was studied. Two groups of 20 adult male and female carcinogenicity and lower formation of DNA adducts (9). Fischer 344 rats consumed 2% green tea or water for 6 This paper explores the effectiveness of green tea in weeks before being administered a single dose of 40 mg/kg enhancing the detoxification of IQ in the F344 rat. The results body weight of [2- 14 C]IQ by oral gavage. Major metabolites show that intake of green tea increases the excretion of Nin 24 h urine samples were separated by high-performance OH-IQ-N-glucuronide, a detoxified metabolite of the proximate liquid chromatography (HPLC), including N-OH-IQ-N- carcinogen, N-OH-IQ, thereby most likely reducing DNA glucuronide, 5-OH-IQ glucuronide and sulfate, IQ sulfa- adduct formation in particular and carcinogenesis in general. mate and IQ itself. The structures of the main metabolites were established by mobility on the HPLC and by mass Materials and methods spectrometry. Sulfate esters and sulfamate were hydrolyzed Chemicals and reagents by 0.1 N HCl for 15 min at 100°C, yielding 5-OH-IQ and [2-14C]IQ (10 mCi/mmol) and unlabeled IQ were purchased from Toronto high levels of IQ. HPLC of the resulting product showed Research Chemicals (Toronto, Canada). DMSO and unlabeled IQ metabolite the N-OH-IQ-N-glucuronide and the 5-OH-IQ glucuronide, standards were available from previous research (2–6,10–12). Purity of all IQ as well as IQ. The male and female rats drinking tea standards were checked by HPLC and found to be over 95%. All other displayed a significantly higher (P < 0.05) excretion of the chemicals were purchased from laboratory supply firms at the highest available grade. two major glucuronides. We conclude that intake of green tea increases the excretion of N-OH-IQ-N-glucuronide, Tea Fresh Lipton Research World Blend of green tea was donated by the Lipton a detoxified metabolite of the proximate carcinogen N- Company (Dr William Franke), Englewood Cliffs, New Jersey, through the OH-IQ. courtesy of the Tea Council of the USA. Green tea (20 g) was dissolved in 1000 ml boiling water and stirred for 5 min. The mixture was filtered to separate the liquid from the leaves. This gave a 2% (w/v) concentration of

Most of the prevailing chronic diseases in the world have an important nutritional component by directly causing a specific disease, enhancing the risk through phenomena of promotion, exerting a beneficial effect in decreasing risk, or preventing the disease. International studies in geographic pathology have shown that a given disease may have vastly different incidence and mortality as a function of residence. Laboratory research in animal models can reproduce fairly accurately what is learned through international research and provide the basis for examining relevant hypotheses and, more importantly, proposed mechanisms of action. Validation of these approaches can be the basis for public-health recommendations and health-promotion activities. Through such techniques, it has been found that regular intake of foods with saturated fats such as meat and certain dairy products raise the risk of coronary heart disease. The total mixed-fat intake is associated with a higher incidence of the nutritionally linked cancers, specifically cancer of the postmenopausal breast, distal colon, prostate, pancreas. ovary, and endometrium. The associated genotoxic carcinogens for several of these cancers are heterocyclic amines, which also play a role in heart-disease causation, and these are produced during the broiling and frying of creatinine-containing foods such as meats. Monounsaturated oils such as olive or canola oil are low-risk fats as shown in animal models and through the observation that the incidence of specific diseases is lower in the Mediterranean region, where such oils are customarily used. High salt intake is associated with high blood pressure and with stomach cancer, especially with inadequate intake of potassium from fruits and vegetables and of calcium from certain vegetables and low-fat dairy products. Vegetables, fruits, and soy products are rich in antioxidants that are essential to lower disease risk stemming from reactive oxygen systems in the body. Green and black teas are excellent sources of antioxidants of a polyphenol nature. as is cocoa and some chocolates. Nutritional lifestyles that offer the possibility of a healthy long life can be adopted by most populations in the world.

Diet can play a major role in cancer prevention. The international differences in cancer incidence are largely accounted for by lifestyle practices that include nutrition, exercise, and alcohol and tobacco use. About 50% of cancer incidence and 35% of cancer mortality in the U.S., represented by cancers of the breast, prostate, pancreas, ovary, endometrium, and colon, are associated with Western dietary habits. Cancer of the stomach, currently a major disease in the Far East, relates to distinct, specific nutritional elements such as excessive salt intake. For these cancers, information is available on possible initiating genotoxic factors, promoting elements, and prophylactic agents. In general, the typical diet in the United States contains low levels of the potent carcinogenic agents, heterocyclic amines, formed during the cooking of meats. It provides only about half the potent appropriate fiber intake and is high in calories. About twice as many calories as would be desirable come from fat, certain kinds of which enhance the development of cancers. Other foods with functional properties, such as soy products and tea, can be beneficial. To achieve reduction in risk of certain cancers, diet must be optimized, primarily to reduce caloric intake and the fat component. The latter should be 20% or less of total caloric intake and fiber should be increased to 25-35 g per day for adults. One approach to achieving these goals is the Fiber First Diet, a diet designed around adequate fiber intake from grains, especially cereals, vegetables, legumes, and fruits, which thereby reduces both calorie and fat intake. Such dietary improvements will not only reduce cancer and other chronic disease risks, but will contribute to a healthy life to an advanced age. A corollary benefit is a lower cost of medical care.

Cancer is a dread disease worldwide. Mortality of individuals suffering from cancer is high, despite the current improved methods of precocious detection, surgery and therapy. Prevention of cancer is the recognized goal of many activities in cancer research. This aim was recognized early to involve the bioassay of environmental chemicals or mixtures. The first such study involved application of coal tar to the ear of rabbits, and later on to the skin of mice. Subsequently, laboratory rats were introduced, and hamsters were utilized as a substitute for the unwieldy tests in rabbits. Investigators also became concerned with the mechanisms of carcinogenesis, and more definitive approaches to carcinogen bioassay in laboratory animals, as possible indicators of cancer risk in humans. These tests were expensive and lengthy, and did not serve the important purpose of accurately measuring risk of cancer to humans. Once it was realized that DNA and the genetic apparatus might be a key target, rapid bioassays in bacterial and mammalian cell systems were introduced successfully. Thus, batteries of tests are now available to detect effectively human cancer risks, and provide novel approaches to determine the underlying mechanisms, as a sound basis for cancer prevention.

Detailed multidisciplinary research on the effect of tea and the associated tea polyphenols has led to major advances on the underlying mechanisms. In most studies, green and black tea have similar effects, four of which are reviewed in this paper. 1) Tea polyphenols are powerful antioxidants that may play a role in lowering the oxidation of LDL-cholesterol, with a consequent decreased risk of heart disease, and also diminish the formation of oxidized metabolites of DNA, with an associated lower risk of specific types of cancer. 2) Tea and tea polyphenols selectively induce Phase I and Phase II metabolic enzymes that increase the formation and excretion of detoxified metabolites of carcinogens. 3) Tea lowers the rate of cell replication and thus the growth and development of neoplasms. 4) Tea modifies the intestinal microflora, reducing undesirable bacteria and increasing beneficial bacteria. The accumulated knowledge suggests that regular tea intake by humans might provide an approach to decrease the incidence of and mortality from major chronic diseases.

International research, particularly as part of US/Japan programs, has led to major advances in knowledge of causes of heart disease, stroke, many types of cancer and diabetes, showing that individual lifestyle is associated with these diseases. In Japan, a major health problem is high blood pressure and stroke, and cancer of the stomach, from excessive use of salt and salted, pickled foods, and the relative low intake of protective fruits and vegetables. We identified a likely gastric carcinogen, 2-chloro-4-methylthiobutanoate, in salted, pickled fish. In the Western world, heart disease and cancer of the breast, colon, rectum, prostate, pancreas, ovary and endometrium relate to a nutritional tradition too high in total fat and fried or broiled meats, and too low in fiber, vegetables and fruits. The cooked meats contain genotoxic chemicals, heterocyclic amines, causative elements in heart disease and the nutritionally linked cancers. Decreasing total fat intake, from 40 to 20% of calories and a greater use of starches such as rice, pasta, potatoes and whole grain bread, as well as daily intake of five to nine vegetables and fruits would be beneficial. Adults should consume 2.5 l of fluids per day. Green or black tea and fruit juices have health promoting properties. Regular exercise contributes to good health, and to the avoidance of obesity, a major problem in the USA and of increasing importance in Japan. Avoidance of a risky lifestyle would likely prevent diseases important not only for the individual and his family, but with major impact in lowering medical care costs. Tobacco and cigarette use, particularly on a Western diet, involve a high risk of heart attacks, and cancers of the lung, pancreas, kidney, urinary bladder, and cervix, accounting for 35% of medical care expenditures.

Many laboratory studies and human epidemiological data suggest that most cancer deaths are attributable to lifestyle, including nutritional factors and tobacco and alcohol consumption. Tobacco consumption is causally related to cancer of the lung, mouth, larynx, esophagus, bladder, kidney, and pancreas. Nutrients and non-nutrient dietary components probably account for cancer of the colon, breast, prostate, and stomach. This report is based on literature and our own data pertaining to the role of dietary fat, calories, and fiber in the development of colon and breast cancer. We also discuss the evidence from epidemiological, mechanistic, and preclinical efficacy studies indicating a protective effect of micronutrients, non-nutrients, and certain antioxidants in food against oral and lung cancers. Given the continuing cancer burden and the relatively slow impact of proven cancer treatment strategies in reducing cancer mortality, it is essential to evaluate promising nutrients and non-nutrients in foods as chemopreventive agents in persons at increased risk for cancer. Development of reliable intermediate biomarkers is valuable for clinical chemoprevention intervention trials. The purpose of this report is to provide the reader with plausible approaches to cancer control.

The primary nutritionally linked diseases are coronary heart disease, stroke, and cancers of the stomach, colon, pancreas, prostate, breast, ovary, and endometrium. Dietary fats operate through a promoting mechanism. An S-shaped dose-response curve with a threshold has been demonstrated in models of breast and colon cancer in which the standard Western fat intake of 40% of energy yields a high level of promotion, and reduction of fat to 10% to 20% of energy (the traditional Japanese fat intake) has a low promoting action. In models of breast and colon cancer, saturated fats such as beef fat or lard, and monounsaturated oils, such as olive oil, display only a'weak promoting effect, with the incidence of induced tumors being similar at intake levels of 40% and 10% of energy. On the other hand, the n-6-polyunsaturated oils display a strong promoting effect. Such findings may have a parallel in the low but definitely increasing slope of postmenopausal breast cancer incidence in the past 30 years as the American public decreased saturated fat intake to avoid heart disease and increased use of the n-6-polyunsaturated oils. Mechanisms underlying the cancer-promoting effect in the colon stem from increased hepatic production of bile acids, which are transferred to the intestinal tract via the bile. Ingestion of 40% fat calories yields higher concentrations of bile acids in the colon than lower levels of dietary fat ingestion. Cancer in the mammary gland is promoted through higher concentrations of fats and phospholipids in the gland as well as increased levels of estrogen secondary to production by the ovary and other endocrine tissues that, in turn, affect the generation of pituitary hormones such as prolactin and growth hormone. The n-3-fats, as found in fish and fish oils, have a pronounced inhibitory effect in models of colon and breast cancer, presumably through their shifting of prostaglandin metabolism to the generation of prostaglandins, which lower cell proliferation potential and, thus, decrease promotional effects. The role of dietary fat as a promoter can be modified by other nutritional components. Finally, one of the best pieces of evidence for an enhancing effect of many dietary fats in the nutritionally linked cancers is the current increase in the incidence of these diseases in Japan as the nutritional habits of people in that country become more Westernized. J Am Diet Assoc. 1997;97(suppl):S16-S23 .

Research was performed on the effect of tea, or tea and milk, instead of drinking water, in rat models of cancer in the mammary gland or colon. Solutions of 1.25% (w/v) black tea, or 1.85% (v/v) milk in tea were prepared three times per week. SD rats were given tea beginning at 42 days of age; one group was gavaged 5 mg 7,12-dimethylbenz[a]anthracene (DMBA) at 49 days of age; another group received 8.4 mg 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) twice per week beginning at age 49, then 14 mg twice a week for 4 weeks more. The groups on DMBA were killed 33 weeks later, and those on IQ 39 weeks later. Tea decreased the mammary gland tumor multiplicity and volume, and milk and tea had a greater protective action. Male F344 rats were given two doses of 15 mg/kg azoxymethane (AOM) on weeks 6 and 7, and some groups started on tea, or tea and milk at 5 weeks; one group started on tea 2 days after AOM. Foci of aberrant crypts in the colon were decreased, after 9 weeks, in the groups on tea, or tea and milk during AOM administration, but not after AOM. Thus, tea decreases mammary tumor induction, and the production of foci of aberrant crypts in the colon. Milk potentiates these inhibiting effects.

Aqueous solutions of gallic acid, methyl gallate, catechins, theaflavins and tannic acid were tested for inhibition of the mutagenicity of PhIP in the Salmonella typhimurium TA98 assay with an S9 fraction from the liver of rats induced with alpha-naphthoflavone and phenobarbital. The IC50S were in the 80-250 microM range for the gallated catechins, theaflavins and tannic acid. No inhibition could be found with these compounds when a direct acting mutagen was used. This indicates that the anti-mutagenic properties of these phenolic compounds may be due to their inhibition of the cytochrome P-450 enzymes.

Cancers of many types are major chronic diseases with a high fatality rate and a high cost to society. In the USA, the Delaney Clause was implemented in 1958 because the public believed that many cancers stem from food additives and food contaminants. In the intervening years, research has provided key information about the mechanisms of carcinogenesis and demonstrated that there are two major classes of carcinogens, genotoxic and non-genotoxic. Two case reports are presented, of sodium saccharin and ethylenebisdithiocarbamates that were banned based on the Delaney Clause in an unjustified manner, based on the underlying mechanisms not relevant for non-genotoxic carcinogens. Also, the causes of major cancers have been discovered. Most cancers are associated with lifestyle, specifically tobacco and excessive alcohol use, inappropriate nutritional traditions, and lack of exercise. These lifestyle components involve now known genotoxic carcinogens and importantly, non-genotoxic carcinogens. The effect of non-genotoxic carcinogens is highly dose dependent and also reversible upon lowering the dose below a threshold. Thus, it is quite possible to lower human cancer risk, and also the risk of related chronic diseases such as coronary heart disease, hypertension and stroke, adult on-set diabetes, by proper lifestyle adjustments. Clearly, the Delaney Clause plays no role in disease prevention.

The Delaney Clause has been part of the US Food, Drug and Cosmetic laws since it was enacted by the Congress in 1958. It states that no cancer-causing agent, as demonstrated in humans or animals, shall be deliberately added to, or found as a contaminant in food. The FDA was charged with enforcing this Clause. Other agencies such as EPA used similar approaches with the avowed aim to prevent cancer. Legal cases have been brought against Agencies who failed to comply with this law to the letter. Since 1958, research has elucidated the main mechanisms whereby chemicals cause cancer. The leading causative factors of the major human cancers are basically known, and include smoking and tobacco use, excessive alcohol, and common dietary practices and nutritional traditions. The etiology of lymphomas, leukemias and cervical cancer may be viruses. The Delaney Clause was designed to protect against these many cancer types. It was based on the hypothesis held in the 1950s that human cancers are due to environmental chemicals. This is clearly not true for the great majority of cancers and therefore, the Delaney Clause as framed has not saved any lives, is obsolete, and should be eliminated.

Summary The document “Risk Assessment of Carcinogens in Food with Special Consideration of Non-Genotoxic Carcinogens” was produced by the International Federation of Societies of Toxicologic Pathologists on the occasion of its triannual meeting in Tours, France, April 23–26, 1995. Subsequently, it was endorsed by the North American Society of Toxicologic Pathologists at its annual meeting in San Diego, CA, USA, June 11–15, 1995. This document was written to address up-to-date risk assessment of carcinogens and anachronisms in the Delaney Clause of the US Federal Food, Drug and Cosmetic Act which have become evident since its enactment in 1958. In the intervening years, major progress has been made in understanding mechanisms of cancer induction and in recognizing causes of human cancer. The Clause in conjunction with its present legal interpretation and implementation does not provide for rational, scientific evaluation of carcinogens. It ignores the fact that the diverse mechanisms now known to underlie cancer increases in rodents exposed to high doses of chemicals are often inapplicable to man. In this regard, current evaluation of chemicals based on the tenents of the Delaney Clause is irrational in many cases. The document presents several examples of chemicals to which humans may be exposed through food and which illustrate the need for science-based risk assessment. Appropriate risk assessment methods are available to provide assurance of negligible risk, and accordingly, it is recommended that the Delaney Clause be rescinded as it has outlived its usefulness. This will enable US governmental agencies to regulate the use of chemicals in foods by using appropriate current scientific methods on a case by case basis within the context of other relevant legislation.

Standard black and green tea extracts have been known to inhibit mutagenicity caused by PhIP, in the Salmonella typhimurium TA98 assay containing S9 fraction from the liver of rats induced with alpha-naphthoflavone and phenobarbital. Breeding and selection programs for high yielding tea clones have successfully increased yields in many tea producing areas. Six clonal teas and three seedling teas were obtained from a tea producing area in Southern Africa. Standard black and green teas were used as controls. Dose-dependent inhibition of the bacterial mutagenicity elicited by two concentrations of PhIP was found in the extracts of all the teas tested. This indicates that the clonal teas have not lost their anti-mutagenic properties. Small differences were found amongst the clonal teas in their ability to inhibit mutagenicity. This indicates that it may be possible to enhance this trait in future breeding and selection programs.

The APC gene plays a major role in human colon carcinogenesis. We determined the genomic structure of the rat Apc gene, and we analyzed mutations in colon tumors induced in F344 rats by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), potent carcinogens contained in ordinary daily human food. Of eight PhIP-induced tumors, one tumor had two Apc mutations, two tumors had a mutation with loss of the normal allele, and one had a mutation. Two of the above five mutations were at nucleotide 1903, one at 2605, and two at 4237, all being a deletion of a guanine base at the 5'-GGGA-3' site and resulting in truncation of the APC protein. Of 13 IQ-induced tumors, 2 had an Apc mutation with loss of the normal allele. The two mutations were a missense mutation (T-->C) at nucleotide 1567 and a nonsense mutation (C-->T) at 2761. Alteration of the Apc gene was shown to play a more important role in PhIP-induced than in IQ-induced rat colon carcinogenesis. PhIP-induced tumors are characterized by their specific and unique mutation, which may be useful for mutational fingerprinting of human cancers.

We investigated the modulation by dietary corn oil and calcium levels of carcinogenesis by heterocyclic amines (HCA), a new class of important carcinogens in the human nutritional environment, since they are formed during cooking. Two approaches involved (i) a chronic bioassay in male and female F344 rats, and (ii) an abbreviated test, the induction of foci of aberrant crypts in the colon in male F344 rats. One typical HCA, 2-amino-3-methylimidazo [4,5-f]quinoline (IQ) was fed at 75 ppm for 12 months to male and female rats that were held three and six months longer, respectively, on control diets. Neoplasms were induced in the Zymbal gland, skin (predominantly in male rats), liver, mammary and preputial glands, colon, and lung. Diets with 23.5% corn oil increased carcinomas in the liver in males, and in the mammary gland in females, compared with a 5% corn oil diet. Males on the low-fat diet had more cancers in the lip, and females had more ear duct cancers, than did rats on the high-fat diet. Another HCA, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), fed at 400 ppm for nine weeks induced foci of aberrant crypts in the lower intestinal tract of male F344 rats. There were significantly more aberrant crypts on the high-fat than on the low-fat diet. On the low-fat diet, there were fewer aberrant crypts on the higher calcium level. Thus, dietary fat modulates the carcinogenic action of HCA food carcinogens in specific organs of male and female F344 rats. Also, both fat and calcium affected the induction of aberrant crypts in the distal intestinal tract of male F344 rats.

Background Compounds formed on the surface of fried or grilled meat and fish may be associated with increased risk of colon cancer. Normal intestinal bacteria can convert one of these compounds, 2-amino-3-methyl-3H-imidazo[4,5-f]quinoline (IQ), to the 7-hydroxy metabolite, 2-amino-3,6-dihydro-3-methyl-7H-imidazolo[4,5-f]quinolin-7-o ne (7-OHIQ), a direct-acting mutagen. Purpose We studied the genotoxicity and carcinogenicity of 7-OHIQ to determine if it is responsible for the colon-specific activity of IQ. Methods The effects of pure, synthetic 7-OHIQ on DNA were evaluated in the Ames Salmonella typhimurium TA98 test, with and without an induced rat liver S9 fraction, and in the Williams DNA repair test using freshly explanted rat hepatocytes. 7-OHIQ was also subjected to an in vivo bioassay for 21 months by long-term intrarectal infusion in male F344 rats, using IQ and N-nitrosomethylurea (NMU) given intrarectally as positive tumor-producing controls. The standard NIH-07 rodent diet was supplemented with 15% corn oil to maximize any effect of the infused materials on the colon. A parallel bioassay involved intraperitoneal injection of 7-OHIQ in newborn mice, followed by dietary administration from week 11 to week 67. Again, IQ and NMU were used as positive controls. Results We confirmed that 7-OHIQ is a direct-acting mutagen in the Ames test, with added S9 liver fraction giving higher mutagenicity. 7-OHIQ was negative in the Williams test, whereas IQ was positive. 7-OHIQ did not induce colon cancer in rats, and in the newborn mouse test it produced only a low incidence of liver neoplasms. Conclusions 7-OHIQ is not genotoxic, for to be so classified it must be definitely positive in both the Ames and Williams tests; moreover, it is not carcinogenic, in marked contrast to IQ and NMU.

The purpose of this study was to determine the carcinogenic effect in male rats of a single i.v. injection of N-methyl-N-nitrosourea (MNU) after sequential treatment with cyproterone acetate (for 21 days) and testosterone propionate (for 3 days). This treatment has previously been shown to induce carcinomas of the prostate and other male accessory sex glands. A wide spectrum of non-melanoma skin tumors was found in 38-48% of Wistar (Cpb:WU) rats given this sequential treatment, but only in 5% of rats that received only MNU. Castration long and, particularly, early after MNU markedly reduced this skin tumor response to a 10-13% incidence. The skin tumorigenic efficacy of MNU was dependent on the time between the start of the testosterone propionate treatment and carcinogen administration: MNU injection after 48-50 or 60-63 h induced skin tumors in 17-21% of Wistar rats, whereas injection after 72-74 h induced a 48% incidence. The Fischer F344 and Sprague-Dawley strains were not very sensitive to induction of skin tumors by this approach. Thyroid follicular cell tumors were also induced by MNU only after the hormonal pretreatment, and their induction was influenced by the time of MNU injection as well. The time of MNU injection and rat strain used did not significantly influence the induction of sebaceous-squamous neoplasms of the ear-duct/Zymbal's glands or other tumors. These data indicate that endogenous androgens are critically involved in the later stages of rat skin tumorigenesis and suggest that androgen-induced cell proliferation influences the initiation stage of this process and, possibly, of thyroid tumorigenesis.

International, multidisciplinary biomedical research in the field of nutrition and cancer has led to the recognition that the traditional Western total fat intake of about 40% calories constitutes a major risk factor for cancer of the postmenopausal breast, distal colon, pancreas, ovary, endometrium, and probably prostate. The underlying complex mechanisms have been sketched out, justifying public health promotion activities involving reduction of fat intake to 20 to 25% of calories, facilitated by the deliberate marketing of low-fat products by the food industries.

Neoplasia in fish can result from contamination of waters with carcinogens and promoters. Cancer in fish, therefore, is a possible indicator of cancer risk to man and serves as a guide to the need for preventive approaches involving improved means of waste disposal and environmental hygiene. Moreover, cancer in fish indicates that this important food source may be contaminated. Detection of genotoxic carcinogens to which fish are exposed can be achieved quickly and efficiently by carefully selected batteries of complementary in vitro and in vivo bioassays. One such battery consists of the Ames test, a reverse mutation assay in prokaryotic Salmonella typhimurium, and the Williams test, involving DNA repair in freshly explanted metabolically highly competent liver cells from diverse species, including humans. Determination of DNA-carcinogen adducts by varied techniques, including 32P-postlabeling, as well as DNA breakage, mammalian cell mutagenicity, chromosome aberrations, sister chromatid exchange, or cell transformation represent additional approaches, each with its own advantages and disadvantages. More research is needed on systems to apprehend neoplasm promoters, but tests to determine interruption of intercellular communications through gap junctions appear promising. Other approaches rely on measurement of enzymes such as ornithine decarboxylase and protein kinase C. Approaches to the definition of risk to fish or humans require characterization of the genotoxic or nongenotoxic properties of a chemical, relative potency data obtained in select, limited rodent bioassays, and knowledge of prevailing environmental concentrations of specific carcinogens.

Fecapentaenes, a class of direct-acting bacterial mutagens, have been isolated from the feces and intestinal tract of humans on a Western meat-containing diet. Two bioassays to test pure fecapentaene-12 (FP-12) for carcinogenicity were performed. FP-12 in dimethylsulfoxide (DMSO) solution was injected i.p. into newborn ICR/MA mice on days 1, 3, 7, 10, 14 and 21. The mice killed after 21 months had neoplasms in liver, lung, glandular stomach and subcutaneous fibrosarcoma. Intrarectal (i.r.) infusion of FP-12 in an aqueous vehicle into male F344 rats for 71 weeks, and killing the rats after 21 weeks more, displayed no evidence of neoplasia associated with FP-12 exposure. The positive control, N-nitrosomethylurea (NMU), given i.r. as 4 2-mg doses in 2 weeks, as expected, yielded multiple colonic neoplasms in less than 11 months. Fecapentaene may exert its effect in bacteria and in newborn mice through the generation of hydroxy radicals. However, adult rodent and human colon may have adequate biochemical defense mechanisms against low level, even continuous exposures to chemicals like FP-12, and thus be at low risk of neoplasia, as was found.

Ernst L. Wynder is internationally known for his important discoveries in the field of human chronic disease causation, that is the underlying mechanisms, studied in various animal models, as a foundation for recommendations on the prevention of these diseases. These include coronary heart disease, and the main human cancers including cancer of the lung, caused by traditional smoking habits, and the nutritionally linked cancers, namely cancer of the breast, prostate, colon, pancreas, and urinary bladder. Much of this research was performed in a chronic disease prevention institution—created by Dr. Wynder—the American Health Foundation. There were outreach programs to educate people about proper lifestyles to secure disease prevention, including beginning health education in children.

A program of research was established on the question whether melatonin played a chemopreventive role in the development of foci of aberrant crypts in the intestinal tract of male rats. Male F344 rats were injected i.p. with an aqueous solution of 15 mg/kg azoxymethane (AOM) on day 50 and day 57, and a group was also injected i.p with 0.5 mg melatonin in 0.5 ml of 10% ethanol solution 5 times per week beginning at age 47 days. Foci and multiplicity of aberrant crypts were determined after 8 weeks. These groups of animals were kept in light daily from 4∶30 to 16∶30.In the group receiving AOM and the melatonin injections, there were fewer foci of aberrant crypts in the colon and the average number of crypts was lower after 8 weeks, compared to the group on AOM alone.Melatonin inhibited the formation of foci of aberrant crypts in the descending colon of rats. Also, it reduced the number of aberrant crypts per focus from foci with 3 and more aberrant crypts.

The modulation by dietary fat levels of intestine carcinogenesis is well documented. New developments suggest that calcium ions may also play a role. A rapid bioassay, the induction of foci of aberrant crypts in the colon, was used to explore the interaction between dietary fat and calcium. Male F344 rats 6 weeks of age were placed on diets containing 5 or 20% corn oil, and 0.04 or 0.32% calcium ion, as calcium lactate. Each dietary group was fed 400 ppm 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhlP), and negative controls received the diets alone. A positive control group was given 2 mg N-nitrosomethylurea (NMU) intrarectally four times in a 2-week period. All rats were killed after 9 weeks. The intestinal tract was rinsed with Krebs-Ringer buffer. After staining a 6-cm segment of the descending colon and rectum with 0.2% methylene blue, foci of aberrant crypts were evaluated microscopically. With PhlP as a carcinogen, the rats on a high-fat, low-calcium level had more foci of aberrant crypts than animals on a low-fat level. With the higher calcium level, there were fewer foci and aberrant crypts, but the effect of fat was still significant. With NMU and a low-calcium level, the effect of fat level was evident. However, with the higher calcium intake, there were considerably more foci of aberrant crypts than on the low-calcium level, and the effect of the dietary fat level was not obvious.(ABSTRACT TRUNCATED AT 250 WORDS)

International research through epidemiological techniques has provided information on risk factors and preventive approaches in chronic disease. Causation complementing this base of knowledge with laboratory research on associated markers for each disease has outlined the possible mechanisms whereby risk factors and preventive conditions operate. Furthermore, laboratory research in animal models and cell cultures has expanded the appropriate elements associated with each condition. Individuals in the Mediterranean area present with a lower risk of several important chronic diseases, including coronary heart disease and a number of types of cancer associated with nutritional traditions, such as breast, colon, and prostate cancer. Vegetables and fruits in general and cooked tomatoes, together with olive oil, appear to be the nutritional traditions that account for this lower risk. These results lead to public health recommendations to consume more vegetables and, especially, cooked tomatoes with olive oil.

Powerful mutagens are formed during the broiling or frying of meat. These mutagens cause specific cancers in animal models, and epidemiological studies suggest that they increase the risk of breast and colon cancer. It is important, therefore, to inhibit the formation of these mutagens. Application of tea polyphenols, polyphenon 60® from green tea, and polyphenon B® from black tea, to both surfaces of ground beef before cooking inhibits the formation of the mutagens in a dose-related fashion. This procedure is simple and effective, and utilizes inexpensive tea, a product that deserves consideration for practical use.

We have explored the causes of the major chronic diseases prevailing in the world and the relevant mechanisms as a sound basis for recommendations for their prevention. Research shows that the cocoa bean, and tasty products derived from the cocoa bean such as chocolate, and the beverage cocoa, popular with many people worldwide, is rich in specific antioxidants, with the basic structure of catechins and epicatechin, and especially the polymers procyanidins, polyphenols similar to those found in vegetables and tea. Metabolic epidemiological studies indicate that regular intake of such products increases the plasma level of antioxidants, a desirable attribute as a defense against reactive oxygen species (ROS). The antioxidants in cocoa can prevent the oxidation of LDL-cholesterol, related to the mechanism of protection in heart disease. Likewise, a few studies show that ROS associated with the carcinogenic processes is also inhibited, although there have not been many studies on a possible lower risk of various types of cancer either in humans or in animal models consuming cocoa butter or chocolates. Based on the knowledge acquired thus far, it would seem reasonable to suggest inhibition of the several phases of the complex processes leading to cancer, as a function of quantitative intake of antioxidants, including those from cocoa and chocolates. Cocoa and chocolate also contain fats from cocoa butter. These are mainly stearic triglycerides (C18:0) that are less well absorbed than other fats, and are excreted in the feces. Thus, cocoa butter is less bioavailable and has minimal effect on serum cholesterol.

The transformation of normal cells by DNA reactive, genotoxic carcinogens and the growth promotion and development of mutated cells by enhancing factors is involved in the overall basic mechanism of cancer induction. Thus, discrimination between genotoxic carcinogens and nongenotoxic chemicals is essential. The dose-response curves, reversibility, and organ-and species specificity are distinct. Genotoxic carcinogens are mutagenic, form DNA adducts, induce DNA repair, and form hydroxy radicals and inappropriate peroxidation reactions that antioxidants such as those in vegetables, fruits, and tea can decrease. In contrast, promoters do not form DNA adducts, but raise cell duplication rates, among other attributes. In the USA, about 35% of known cancers are associated with tobacco use and about 55% with inappropriate nutritional habits. Cancer induction can be decreased by avoiding the formation of carcinogens, reducing their metabolic activation, or increasing their detoxification. Excessive dietary salt, and heterocyclic arylamines formed in cooking of meats or fish, and high intake of 40% of calories in fats are health risks, but vegetables, fruits, tea, soy products, and fibers are protective. We review nutritional factors involved in cancer and chronic disease causation and prevention.

Much progress has been achieved by exploring the causes of the main human cancers and of cardiovascular and cerebrovascular diseases. Even more important has been the knowledge acquired about the mechanisms underlying the development of these diseases. In many parts of the world, particularly in the West, the major cancers associated with dietary habits involve the postmenopausal breast, distal colon, prostate, pancreas, ovary, and endometrium. Current evidence suggests that the genotoxic carcinogens for all but the last 2 of these diseases stem from the traditional intake of fried and broiled foods such as meats. The surface of these foods contains a class of powerful mutagens, heterocyclic amines, which are carcinogenic to the target organs in animal models. Fish-eating populations have lower incidences of heart disease and of many types of cancers than do other populations, which may be the result of the n-3 polyunsaturated oils found in fish. Among other dietary practices that may reduce the risk of cancer and cardiovascular disease are consuming 5-9 servings of fruits and vegetables daily, which provides antioxidants such as quercetin and isothiocyanates; having a high fiber intake, including bran cereal; and drinking 1.5-2.5 L of fluids daily. Tea polyphenols found in black and green tea may have a protective effect against heart disease and some cancers. Concentrates of such desirable products have been made available in pill form to complement health-promoting personal lifestyles. Biomedical research funded by The National Institutes of Health and organizations such as the American Cancer Society has produced sound results that could lead to prevention of chronic disease. The public must heed this information to achieve long-term health.