Heterocyclic Amines

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  2. August 2, 2012 12:56 pm



People nowadays are becoming much more health conscious and many are willing to make fundamental changes in their diet in order to obtain greater health and longevity. Unfortunately much of the advice available is conflicting. Many self styled nutritionists make pronouncements concerning diet with little or no basis of scientific evidence. It is therefore important that careful scientific studies are carried out so that sound advice can be given.


Humans are classed as omnivores – in other words, they are capable of consuming and digesting and surviving on a variety of types of diet. The species is omnivorous but the individual does not have to be. All that nature is concerned with is survival through the reproductive years. But people now desire their health to last into ripe old age. The question therefore is what type of diet should they be advised to eat.


Throughout history, the majority of mankind has subsisted on vegetables, fruits and grains with meat being only an occasional luxury on feast days or other special occasions. It is only in recent times that meat has been thought of as desirable from a health point of view. And it is only really in this century and only in industrialised countries that meat has come to be regarded as a daily necessity for good health. Despite this notion having no scientific basis, it became firmly established in the West in the period between the two world wars. Indeed proponents of vegetarianism like George Bernard Shaw were ridiculed by the medical profession. 


Then, in the 1950s and 1960s, atherosclerosis and various cancers became much more common and population studies started to show their relationship with diet. We now have good epidemiological evidence to advise people to eat less meat and more fruit and vegetables but our understanding of why this should be is still fairly rudimentary.


In September 1997, the World Cancer Research Fund, in association with the American Institute for Cancer Research, issued a report entitled "Food, Nutrition and the Prevention of Cancer: a Global Perspective." The 650-page report was based on examination of 4,000 studies of diet and cancer by an international panel of 15 scientists supported by 100 reviewers. The Report considered that "66-75% of colon cancers...... are preventable by a healthy diet."


There have been a number of good studies on the biochemical reasons why fruit and vegetables promote health. "The science base is very strong that fruits and vegetables are protective for all the gastrointestinal cancers and all the smoking-related cancers," says Tim Byers, Professor of Preventive Medicine at the University of Colorado Health Sciences Center in Denver. That includes cancers of the lung, colon, stomach, mouth, larynx, oesophagus, and bladder. And a recent study found that lycopene - a carotenoid in tomatoes and tomato sauce - may protect against prostate cancer. It is not clear how fruits and vegetables may cut cancer risk. It could be their phytochemicals - things like carotenoids, vitamins C and E, selenium, indoles, isothiocyanates, flavonoids, phenols, and limonene. "Men who eat red meat as a main dish five or more times a week have four times the risk of colon cancer of men who eat red meat less than once a month," says Edward Giovannucci of Harvard Medical School.


Heavy-red-meat eaters were also twice as likely to get prostate cancer in his study of 50,000 male health professionals. That's just one study. Looking at others, says Lawrence Kushi of the University of Minnesota, "the evidence is quite consistent that red meat is associated with a higher risk of colon - and possibly prostate - cancer." There is also evidence that women with high intakes of fried meat have an elevated risk of breast cancer and other female hormone related cancers (1). It is evident that mutagenic compounds in fried meat foods may exert their effects all over the body and not only in the tissues lining the digestive tract (2).


How red meat may promote tumours is still a question. Even lean red meat seems to increase the risk of colon cancer. "It could be the carcinogens created when meat is cooked or meat's highly available iron, or something else in meat," speculates Dr. Walter Willett of the Harvard School of Public Health. As early as 1981 Doll and Peto suggested that 90% of colon cancer cases in the US might be prevented by changes in the diet but the specific factors that are responsible have still not been fully established (3). The prevailing hypotheses have been that colorectal cancer risk is increased by dietary fat and decreased by dietary fibre intake(4). However, although carcinogenic heterocyclic amines are formed during frying of meat, especially at high temperatures (5), few epidemiological analytical studies on diet and colorectal cancer considered the method of cooking, and, particularly, the preference for browning of the meat surface, until the early 1990s (6).


The American Institute for Cancer Research, the third-largest cancer charity in the United States, sponsored a panel of 15 scientists, who analysed about 4,500 scientific studies and papers on the relationship between cancer and diet and other "lifestyle" behaviours, such as exercise and alcohol consumption. The report contains no new research. Titled "Food, Nutrition and the Prevention of Cancer: A Global Perspective," the 670-page document estimates that up to 40 percent of cancer world-wide could be prevented if people ate a plant-based, low-fat diet rich in a variety of fruits and vegetables, exercised regularly and maintained a healthful weight.


Researchers at the National Cancer Institute, a division of the National Institutes of Health, are more reluctant to commit to numbers when estimating the role of diet in preventing cancer. "There is strong evidence that diet is associated with cancer risk and mortality," said Dr. Carolyn Clifford, chief of NCI's diet and cancer branch. "But we don't have enough information to put a quantity on the precise role of diet." Still, NCI has several dietary modification trials to study the link between diet and cancer.


And it was NCI - in conjunction with the Produce for Better Health Foundation - that in 1991 launched the "5 a Day Campaign," a marketing effort to encourage Americans to eat at least five fruits and vegetable servings per day to reduce the risk of cancer.


 There are now 10.3 million new cases of cancer each year around the world. The number is expected to rise to 14.7 million a year by 2020.


In addition to fibre, vitamin C, and folic acid, fruits and vegetables offer the antioxidant properties of beta-carotene and other carotenoids, and phytochemicals - such as allicin in garlic and onions, isoflavones in soybeans, sulforaphane in broccoli, and indoles in the cabbage family - which stimulate enzymes in our bodies that detoxify cancer-causing substances.


Attempts have been made to isolate and study various of the chemicals contained in meat, some with negative results. These chemicals include Benzopyrene (7), Methyl Cholanthrene (8) and Nitrosamines. The present study discusses the health risks of Heterocyclic Amines (HCAs). HCAs are amongst the most potent mutagens assessed in the Salmonella mutagenicity assay (Ames test) (9).


Cooking of food is a process unique to humans. It enhances the taste and digestibility of food so much that its beneficial nature is taken for granted; however, it induces profound changes in all types of foods. That these changes may be of concern to human health was established by research in the 1970s and 1980s (10).


Common cooking procedures such as broiling, frying, barbecuing (flame-grilling), heat processing and pyrolysis of protein-rich foods induce the formation of potent mutagenic and carcinogenic HCAs. Risk assessment for the human population consuming these compounds requires the integration of knowledge of dosimetry, metabolism, carcinogenic potency, and epidemiology. When this integration is done in even a preliminary way, the range of risk for an individual from these compounds is enormous. Exposure contributes a range of 200-fold or more and metabolism and DNA repair differences among individuals could easily be an additional 10-fold between individuals. This indicates that differences in human cancer risk for HCAs could range more than a thousandfold between individuals based on exposure and genetic susceptibility. (11)


(HCAs), in particular the aminoimidazoazarenes (AIAs) such as 2-amino-2-methylimidazolo[4,5-f]quinolone (IQ), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), are potent genotoxic chemicals which are formed at part per billion levels when meat is cooked. Using assays based on gas chromatography/mass spectrometry with stable isotope labelled analogues as internal standards workers have demonstrated that MeIQx and PhIP are efficiently absorbed into the systemic circulation after ingestion of fried beef. Using a potent and selective inhibitor of human CYP1A2, furafylline, they showed that N-hydroxylation catalysed by this enzyme is the major pathway of metabolism of MeIQx and PhIP and is solely responsible for their oxidation to mutagenic species. This is in contrast to the situation in laboratory animals in which both activation by N-hydroxylation and deactivation by C-oxidation occurs. When furafylline was administered to human volunteers before ingestion of fried beef, they showed that > 90% of MeIQx and approximately 70% of PhIP elimination could be inhibited, demonstrating the extent to which activation occurred in man. MeIQx is a very powerful mutagen in bacterial assays whereas PhIP is a more potent mammalian cell mutagen. Using a mammalian cell target gene, hprt, they showed that PhIP induces a characteristic mutational 'fingerprint' which is identical to that detected in the Apc gene of 5/8 colonic tumours induced by PhIP in rats. These studies support a biological association between HCA exposure and diet-related tumours but emphasise that information obtained from animal studies does not always reflect the situation in humans. (12)

Figure: Chemical structure of PhIP

chemical structure


Various kinds of mutagenic and carcinogenic HCAs are produced by heating protein-rich foods, such as meat and fish. It has been postulated that thermal interaction of sugars and nitrogenous compounds (e.g. amino acids , creatinine) results in dehydration, condensation, cyclisation and polymerisation of meat surface contents and the formation of heterocyclic compounds (13). Mutagenicity can be assessed , after extraction of the basic fraction of cooked meat samples, by using Salmonella typhimurium strain TA1538 with added rat liver S-9 homogenate. A close association between cooking temperature and mutagenic content in meats has been demonstrated. The surface temperature seems to be particularly important. Barbecuing and roasting appear less harmful than grilling (American broiling) or frying. Boiling, stewing and microwaving result in little or no mutagenicity. Although benzo[a]pyrene and other polycyclic aromatic hydrocarbons are produced during barbecuing (7), non-polar compounds will be removed by the extraction procedure and are unlikely to contribute substantially to basic fraction mutagenicity.



Cooking methods such as grilling and frying which encourage water loss lead to considerable mutagen production, associated with the intensity of browning. Direct heat transfer between the cooking medium and the meat surface appears to be a major factor contributing to the considerable amount of mutagen production in fried meats. The role of fat in mutagen production has been examined and it has been suggested that fats are sources of intermediates for mutagen production. However, most researchers agree that fat is not a reactant in mutagen formation but is involved in more effective heat conduction.



Similar levels of mutagenicity occur in beef as in lamb (14).


To evaluate the risk of HCAs in terms of human cancer development, exposure levels must be measured. A group of workers in Tokyo therefore analysed their amounts in various kinds of cooked foods and in urine samples of healthy volunteers living in Tokyo. Based on the obtained quantitative data, daily exposure levels to MeIQx and PhIP were calculated to be 0.3-3.9 and 0.005-0.3 microgram per person, respectively. Moreover, human DNA samples were analysed with the 32P-postlabeling method, and colon, rectum and kidney tissues were found to contain an adduct spot corresponding to the standard 5'-pdG-C8-MeIQx by TLC and HPLC, at levels of 14, 18 and 1.8 per 10(10) nucleotides, respectively. The beta-carboline compound, norharman, is produced by heating L-tryptophan, and is known to be present in cooked foods and in cigarette smoke at higher levels than mutagenic and carcinogenic HCAs. While norharman is not itself mutagenic to Salmonella, it does become mutagenic to S. typhimurium TA98 with S9 mix in the presence of non-mutagenic aromatic amines like aniline and o-toluidine. When the workersexamined whether DNA adducts are formed in the DNA of S. typhimurium TA98 by treatment with norharman and aromatic amines using 32P-postlabeling analysis, DNA adduct formation by norharman with aromatic amines was found to be related to the appearance of mutagenicity by norharman with aromatic amines. (15) 


During the cooking of meats, several highly mutagenic heterocyclic amines (HCAs) are produced – at least sixteen have been identified (5a). Many of these have been shown to be carcinogenic in rats and mice (5b). Three HCAs, IQ, MeIQx, and PhIP have been under study for carcinogenicity in cynomolgus monkeys - IQ in particular has been shown to be a potent hepatocarcinogen. Concomitantly, the metabolic processing of these HCAs has been examined. Metabolism studies show that the potent hepatocarcinogenicity of IQ is associated with the in vivo metabolic activation of IQ via N-hydroxylation and the formation of DNA adducts. In monkeys undergoing carcinogen bioassay with IQ, N-hydroxylation was confirmed by the presence of the N-hydroxy-N-glucuronide conjugate of IQ in urine. The N-hydroxylation of IQ appears to be carried out largely by hepatic CYP3A4 and/or CYP2C9/10, and not by CYP1A2, an isoform not expressed in liver of this species. Notably MeIQx is poorly activated in Cynomolgus monkeys and lacks the potency of IQ to induce hepatocellular carcinoma after a 5-year dosing period. The poor activation of MeIQx appears to be due to the lack of constitutive expression of CYP1A2 and an inability of other cytochromes P450, such as CYP3A4 and CYP2C9/10, to N-hydroxylate the quinoxalines. MeIQx is detoxified in monkeys largely by conjugation with glucuronide at the N-1 position. Although the carcinogenicity of PhIP is not yet known, the metabolic data suggest that PhIP will be carcinogenic in this species. PhIP is metabolically activated in vivo in monkeys by N-hydroxylation, as discerned by the presence of the N-hydroxy-N-glucuronide conjugate in urine, bile, and plasma. PhIP also produces DNA adducts that are widely distributed in tissues. The results from these studies support the importance of N-hydroxylation in the carcinogenicity of HCAs in non-human primates and by analogy, the importance of this metabolic activation step in the possible carcinogenicity of dietary HCAs in humans. (16)


Epidemiological studies of colon cancer using crude surrogates for HCAs exposure (e.g., doneness of meat) have produced inconsistent results. To improve exposure assessment of HCAs, R. Sinha and co-workers developed a database of HCA concentrations in commonly consumed meat items cooked by various techniques and degrees of doneness. HCA type and level are dependent on multiple factors, including type of meat e.g., steak, chicken, bacon), cooking technique (with substantial variability present even within high temperature cooking methods), place of preparation (e.g., home, restaurant, or 'fast-food' restaurant), as well as the degree of doneness and surface browning/charring. They developed a questionnaire with meat photographs linked to this database. In addition, they carried out a metabolic study of HCA exposure among 66 subjects to identify biomarkers of HCA exposure which may be useful in epidemiologic studies. These studies should help clarify the role of HCAs in human carcinogenesis, and eventually allow an estimation of the cancer burden in the population attributable to these compounds. (17)


In this field, animals studies are of doubtful value, human epidemiological studies being preferred. Molecular epidemiology offers an improved means to assess cancer risk in humans directly and provides a laboratory in which to test mechanistic hypotheses concerning environmental carcinogenesis(18). But, of course, many studies have been done. And they also point to the dangers of meat. Feeding experiments in mice have yielded tumours of the liver, lung and forestomach (19), (20), and leukaemias/lymphomas (21). In rats, tumours of the liver, small and large intestines, zymbal gland and skin have been produced (22). Feeding experiments in rodents and non-human primates have demonstrated DNA adducts in possible target organs including the liver, kidney, colon, stomach and bladder (23). These studies do show that human meat eating populations are routinely ingesting appreciable quantities of carcinogens. This provides a more satisfactory explanation for the internationally recognised correlation between per capita meat consumption and rates of colon cancer (24), a relation that had previously been ascribed to animal fat or animal protein.


Host factors probably play an important role. AIAs require metabolic activation to be carcinogenic and, in the human and other mammals, this activation appears to be the result of the action of a specific P450 enzyme with unknown interindividual differences (25). It is possible that genetic differences in AIA metabolism and excretion could influence the risk associated with a given level of cooked meat ingestion. Probably populations with low per capita meat consumption are at low risk for colorectal cancer and (perhaps some other tumours) partly because of low AIA uptake.



For omnivorous individuals in industrialised Western countries risk of cancer might relate to cooking practices, as well as to metabolic and excretory patterns under genetic control. This genetic component might contribute to the familial aggregations of colorectal neoplasia that have been reported (26).


There is already sufficient evidence for Governments to warn people of the dangers of meat eating, especially of eating charred meat. When this advice is added to previous advice not to eat undercooked meats because of the risk of infectious disease, it is no wonder that more people are choosing vegetarian alternatives.


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February 2nd 1998.


November 17, 1998  WASHINGTON (CNN) 


Women who consistently eat very well-done beef or bacon may have a higher risk of developing breast cancer than women who eat those foods cooked rare or medium, according to a report released Tuesday in the Journal of the National Cancer Institute. Researchers from the University of South Carolina gave questionnaires to 273 women who were diagnosed with breast cancer between 1992 and 1994 as well as 657 women who were cancer-free. They found women who routinely ate three meats -- hamburger, beefsteak and bacon -- very well done had a 462 percent greater chance of developing  breast cancer. Women who regularly consumed these meats individually had lower increases in risk for breast cancer. The risk for very well done vs. rare or medium was 50 to 70 percent greater for hamburger and bacon, and 220 percent greater for beefsteak.  "We have found a link between well-done meat and  breast cancer, but we are still not sure of the cause," said researcher Dr. Wei Zheng. "This is just one study. It is too early to jump to a final conclusion."  Researchers think compounds of amino acids and proteins called heterocyclic amines and possibly other compounds formed when meats are cooked at high temperatures, often by frying or grilling, may be the links to the increased risk. The study did not look at consumption of fish and chicken cooked at high temperatures. Study findings showed no association with other dietary factors such as dietary fat and breast cancer. In addition, researchers said they found only a weak association between red meat intake and a higher risk of breast cancer.  The study also took into account family history of breast cancer, hormone replacement therapy and waist-to-hip ratio, which are factors currently associated with an increased risk of breast cancer. In an editorial, critics point out that the results of the study did not demonstrate a strong enough  connection between well-done meats and breast cancer.  It stated women should more about the dangers of undercooked meat and the health risks posed by food poisoning.  Undercooked meat can harbor bacteria such as E. coli and salmonella, the cause of many recent foodborne outbreaks of illness across the United States. Zheng recommended boiling, steaming or baking meat until thoroughly cooked, but not charred.

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