Mayte Sanz Alaejos

Venerando González

Ana María Afonso

Maite Sanz Alaejos

Exposure to heterocyclic aromatic amines from consumption of cooked red meat and effect on human cancer risk -a review

Keywords: Heterocyclic aromatic amines, intake, human cancer risk .manuscriptcentral.com

Exposure to heterocyclic aromatic amines from consumption of cooked red meat and effect on human cancer risk. A review

Introduction

To date, more than twenty heterocyclic aromatic amines (HAAs) have been isolated as potent mutagens in the Ames/Salmonella test, and have been characterized. Table I shows their structures, chemical and abbreviated names.

Insert Table I about here

These HAAs contain from two to five (generally three) condensed aromatic cycles with one or more nitrogen atoms in their ring system and, usually, one exocyclic amino group. In general, they are formed during heating process of organic products containing nitrogenous compounds, mainly proteins. This formation is mainly temperature-dependent and so HAAs are classified into at least two groups due to the formation process. HAAs formed at temperatures between 100 and 300ºC are known as "thermic HAAs", IQ type or aminoimidazoazarenes. The ones formed at higher temperatures, above 300ºC, are known as "pyrolytic HAAs" or non-IQ type. Thermic HAAs are generated from the reaction of free amino acids, creatin(in)e and hexosas. The precursor undergoes further dehydration and cyclization to form the observed pyrrole and pyridine derivatives.

The heterocyclic pyridines and pyrazines formed in the Maillard reaction between hexose and amino acids then undergo further transformation with participation of Strecker aldehydes and creatin(in)e to produce imidazo-quinoxalines, perhaps through free-radical reactions. However, with temperatures about 225-250ºC, these compounds seem to degrade or react with other compounds.

On the other hand, the formation takes place through pyrolytic reactions between amino acids and proteins in the case of the non-IQ type. Pyrolysis occurs at temperatures well above 300ºC, and produces many reactive fragments through radical reactions. These fragments are believed to condense to form new heterocyclic structures, and pyrolytic mutagens might be formed via freeradical reactions.

HAAs are mutagenic not only for bacteria, but also for some mammalian cell systems and can produce chromosomal aberrations and sister chromatid exchanges in cultured cells. Some of them show higher mutagenic activity in bacteria and certain animals than typical mutagens/carcinogens such as benzo{a}pyrene or aflatoxin B 1. HAAs like harman and norharman are not mutagenic, but enhance the genotoxicity of mutagenic HAAs. In 1993, the International Agency for Research on Cancer (IARC, 1993) considered eight of the HAAs tested (MeIQ, MeIQx, PhIP, AαC, MeAαC, Trp-P-1, Trp-P-2 and Glu-P-1) as possible human carcinogens (class 2B) and one (IQ) as a probable human carcinogen (class 2A). More recently, in 2002, IQ was included as reasonably anticipated to be a human carcinogen in the NTP 10 th Report on Carcinogens (NTP 2002). Latterly, MeIQ, MeIQx, and PhIP are also listed in the NTP 11 th Report on Carcinogens (NTP 2004) as reasonably anticipated to be human carcinogens, together with IQ. These results are based on the sufficient evidence of carcinogenicity and supporting genotoxicity data obtained in long-term animal feeding studies. Although epidemiological evidence suggests that consumption of well-done or grilled meat may be associated with increased cancer risk in humans, the data are insufficient to support the conclusion that this risk is due specifically to MeIQ, MeIQx, or to PhIP, present in these foods. Case-control studies show very conflicting results. Other case-control studies suggest that any of MeIQ, MeIQx, or PhIP may increase the risk of certain cancers, but they were not associated with other types of cancers. In relation to IQ, published epidemiology studies provide some indication that human cancer risk is related to consumption of broiled or fried foods that may contain IQ and/or other HAAs (NTP 2004). So, a reduction to the exposure of these compounds has been recommended. In any case, it is important to have reliable data on the content of these HAAs in different types of foods prepared in several ways in order to assess the effects associated with their intake.

The present review collects bibliographic data from the last ten years, on the possible relationship between the intakes of certain foods and cancer risk. This paper belongs to a series of reviews on different aspects of heterocyclic amines, such as a) the occurrence and formation of HAAs during the cooking of food, mainly meat and fish products (Sanz Alaejos and Afonso); b) analytical methods applied to HAAs determination in foods (Sanz Alaejos et al. a); and c) HAAs intake, genetic aspects and human cancer risk (Sanz Alaejos et al. b).

Possible relationships between HAAs and human cancer

Epidemiological evidence appears to imply that an excessive consumption of very well-done red meat (HAAs-containing meat products), may induce certain types of cancer [START_REF] Navarro | Meat cooking habits and risk of colorectal cancer in Córdoba, Argentina[END_REF][START_REF] Nowell | Analysis of total meat intake and exposure to individual heterocyclic amines in a case-control study of colorectal cancer: contribution of metabolic variation to risk[END_REF]. "Red meat" usually refers to beef, veal, pork, mutton, and lamb. The so-called "processed meat" groups ham, cured or smoked meats, cold cuts, charcuterie, and meat-products.

The introduction of the Ames test in 1975 provided a rapid method to isolate potential carcinogens from food on the basis of their mutagenic activity. If comparing the mutagenic activity in meat samples to the one related to the known HAAs, most samples present mutagenic activity that cannot be exclusively associated to the aromatic amines currently identified. This suggests that additional compounds other than HAAs are present in these foods and need to be investigated, [START_REF] Anderson | Dietary intake of heterocyclic amines and benzo(a)pyrene: Associations with pancreatic cancer[END_REF][START_REF] Balbi | Food and risk of bladder cancer: a case-control study in Uruguay[END_REF][START_REF] Jägerstad | Genotoxicity of heat-processed foods[END_REF].

Several epidemiological studies have been performed to test the hypothesis of an association between meat intake and human cancers (Zimmerli et al. 2001). It becomes really difficult to link meat consumption to the HAAs intake in practically all these studies. Many other substances and factors which possibly contribute to the etiologies of these cancers cannot be excluded, e.g. nitrosamines, polycyclic aromatic hydrocarbons (PAHs), acrylamide, high fat or salt intake, physical activity and others [START_REF] Anderson | Dietary intake of heterocyclic amines and benzo(a)pyrene: Associations with pancreatic cancer[END_REF][START_REF] Augustsson | Dietary heterocyclic amines and cancer of the colon, rectum, bladder, and kidney: a population-based study[END_REF][START_REF] Balbi | Food and risk of bladder cancer: a case-control study in Uruguay[END_REF][START_REF] Stefani | Meat intake, heterocyclic amines and risk of breast cancer: a case-control study in Uruguay[END_REF], 1998a, 2001;[START_REF] Gunter | Meat intake, cooking-related mutagens and risk of colorectal adenoma in a sigmoidoscopy-based casecontrol study[END_REF][START_REF] Jägerstad | Genotoxicity of heat-processed foods[END_REF][START_REF] Ji | Dietary factors and the risk of pancreatic cancer. A case-control study in Shanghai, China[END_REF][START_REF] Lemarchand | A case-control study of diet and colorectal cancer in a multiethnic population in Hawaii (United States): lipids and foods of animal origin[END_REF][START_REF] Lemarchand | Well-done red meat, metabolic phenotypes and colorectal cancer in Hawaii[END_REF][START_REF] Murtaugh | Meat consumption patterns and preparation, genetic variants of metabolic enzymes, and their association with rectal cancer in men and women[END_REF][START_REF] Navarro | Meat cooking habits and risk of colorectal cancer in Córdoba, Argentina[END_REF][START_REF] Nowell | Analysis of total meat intake and exposure to individual heterocyclic amines in a case-control study of colorectal cancer: contribution of metabolic variation to risk[END_REF][START_REF] Oreglia | Meat, fat and risk of laryngeal cancer: a case-control study in Uruguay. Oral Oncology 37:141-145. mutagenicity and colorectal adenoma risk[END_REF][START_REF] Probsttensch | Meat preparation and colorectal adenomas in a large sigmoidoscopy-based case-control study in California (United States)[END_REF][START_REF] Singh | Dietary risk factors for colon cancer in a low-risk population[END_REF][START_REF] Sinha | Fried, well-done red meat and risk of lung cancer in women (United States)[END_REF]Sinha et al. , 1999[START_REF] Sinha | Dietary intake of heterocyclic amines, meat-derived mutagenic activity, and the risk of colorectal adenomas[END_REF][START_REF] Sinha | Meat, meat cooking methods and preservation, and risk for colorectal adenoma[END_REF][START_REF] Zheng | Well-done meat intake and the risk of breast cancer[END_REF].

Cancer development attributed to HAAs intake is shown in Table II together with some other dietary factors for comparison purposes.

Insert Table II about here

Table III shows the enhancing and protective effects exerted by dietary components on cancer risk development when associated with consumption of foods potentially containing HAAs.

Usually, odds ratios (OR) are used. Sometimes relative risks (RR) are used, but in some studies no indication is made.

Insert Table III about here

Animal fat intake has been frequently, but inconsistently, associated to colorectal cancer risk. Intakes of total fat, saturated fat (S), and polyunsaturated fat (P) are not related with the risk of this disease. However, an inverse association is found for the P/S ratio. So, this ratio may be an indicator of colorectal cancer [START_REF] Lemarchand | A case-control study of diet and colorectal cancer in a multiethnic population in Hawaii (United States): lipids and foods of animal origin[END_REF]. The fat content of red meat diminishes with the intensity of cooking. Therefore, potential higher risk associated with well-done beef may be counterbalanced with the lower fat content of meat. Red and total meat intakes are associated with strong increases in risk of squamous cell laryngeal cancer. This effect disappears after controlling the total fat intake. On the other hand, total fat intake displays a strong association with risk of laryngeal cancer when red meat is also included. Total fat intake combines its effect multiplicatively with tobacco smoking [START_REF] Oreglia | Meat, fat and risk of laryngeal cancer: a case-control study in Uruguay. Oral Oncology 37:141-145. mutagenicity and colorectal adenoma risk[END_REF]). However, MeIQx consumption was inversely associated with smoking habits in relation to lung cancer. Neither DiMeIQx nor PhIP show an association with smoking categories on lung cancer histology, and probably, are not associated with lung cancer risk [START_REF] Sinha | Dietary heterocyclic amines and the risk of lung cancer among Missouri women[END_REF].

Vitamins C and E, vegetables and fruits are associated with reduced risks of prostate and other cancers in population-based studies [START_REF] Chan | Role of diet in prostate cancer development and progression[END_REF][START_REF] Chiu | Dietary factors and risk of colon cancer in Shanghai (China)[END_REF][START_REF] Pellegrini | Food, nutrients and prostate cancer: a case-control study in Uruguay[END_REF]De Stefani et al. 1998a;[START_REF] Hu | Diet and brain cancer in adults: a case-control study in northeast China[END_REF][START_REF] Vecchia | Attributable risks for colorectal cancer in northern Italy[END_REF][START_REF] Nomura | Casecontrol study of diet and other risk factors for gastric cancer in Hawaii (United States)[END_REF]Sinha et al. 1999). The possible explanation is that vitamins C and E inhibit the nitrosation when nitrosamines or other N-nitroso compounds are present in tissues. Also, fiber and carotene seem to exert a decline in cancer risk [START_REF] Chiu | Dietary factors and risk of colon cancer in Shanghai (China)[END_REF][START_REF] Pellegrini | Food, nutrients and prostate cancer: a case-control study in Uruguay[END_REF][START_REF] Hu | Diet and brain cancer in adults: a case-control study in northeast China[END_REF]Kampman et al. 1995;[START_REF] Vecchia | Attributable risks for colorectal cancer in northern Italy[END_REF][START_REF] Shannon | Relationship of food groups and water intake to colon cancer risk[END_REF][START_REF] Singh | Dietary risk factors for colon cancer in a low-risk population[END_REF][START_REF] Slattery | Diet diversity, diet composition, and risk of colon cancer (United States)[END_REF]. However, in relation to pancreatic cancer, high intakes of grilled/barbecued red meat increase odds ratios, but no significant changes are further detected by adjustment the fruit, vegetables, calories, total fat, or alcohol consumption [START_REF] Anderson | Meat intake and cooking techniques: associations with pancreatic cancer[END_REF]. Although the high meat-low vegetable diet is considered the reference high-risk diet of colorectal cancer, other risk dietary patterns are emerging, such as high intakes of processed meat and refined carbohydrates.

The strong association with refined cereal products is consistent with the hypothesis of a role of hyperinsulinism in colorectal carcinogenesis [START_REF] Mc | Food as risk factors for colorectal cancer: a case-control study in Burgundy (France)[END_REF]. Total dietary diversity is not associated with colon cancer [START_REF] Slattery | Diet diversity, diet composition, and risk of colon cancer (United States)[END_REF]. However, eating a diet with a great diversity of meats, poultry, fish and eggs is associated with a 50% increase in risk among all the men.

Women who eat a diet with a more diversified pattern of vegetables presented, approximately, a 20% lower risk than women who ate a non-diversified diet in vegetables [START_REF] Slattery | Diet diversity, diet composition, and risk of colon cancer (United States)[END_REF]. There are few epidemiological studies in which an exclusive association between HAAs and human cancer can be assumed.

1. Red meat intake: Experts from the World Cancer Research Fund and the American Institute for Cancer Research (WCRF/AICR, 1997) reviewed data on red meat intake and cancer risk. Those experts concluded that a high intake of red meat probably increases the risk of developing colorectal cancer, and possibly increases the risk of pancreas, breast, prostate and kidney cancers.

Some reviews on meat consumption and cancer of the large bowel have been published [START_REF] Norat | Meat consumption and colorectal cancer: a review of epidemiologic evidence[END_REF]Truswell 2002). One of these reviews concludes that the relationship between meats and colorectal cancer looks generally weaker than the "probable" status stated by the WCRF in 1997. Nonetheless, it is possible that certain meats or sausages cooked at very high temperature can imply some risk (Truswell 2002). The hypothesis that consumption of red and processed meat increases colorectal cancer risk is reassessed in a meta-analysis of articles published during 1973-1999. Total meat consumption is not significantly associated with colorectal cancer risk [START_REF] Kato | Prospective study of diet and female colorectal cancer: The New York University women's health study[END_REF][START_REF] Lemarchand | Well-done red meat, metabolic phenotypes and colorectal cancer in Hawaii[END_REF][START_REF] Navarro | Characterization of meat consumption and risk of colorectal cancer in Córdoba, Argentina[END_REF][START_REF] Norat | Meat consumption and colorectal cancer: a review of epidemiologic evidence[END_REF]Sinha et al. 1999[START_REF] Sinha | Meat, meat cooking methods and preservation, and risk for colorectal adenoma[END_REF], but high intake of red meat, and particularly of processed meat, is associated with a moderate increase on colorectal cancer risk in all sexes [START_REF] Lemarchand | Well-done red meat, metabolic phenotypes and colorectal cancer in Hawaii[END_REF][START_REF] Norat | Meat consumption and colorectal cancer: a review of epidemiologic evidence[END_REF]Sinha et al. 1999), or only in men [START_REF] Lemarchand | A case-control study of diet and colorectal cancer in a multiethnic population in Hawaii (United States): lipids and foods of animal origin[END_REF]. Sinha et al. (1999) found a higher risk of colorectal adenoma mostly confined to intake of well-done/very welldone red meat and meats cooked at high temperature, such as grilled and, possibly, fried red meat.

However, other authors [START_REF] Navarro | Characterization of meat consumption and risk of colorectal cancer in Córdoba, Argentina[END_REF] do not find any relation between high consumption of total-, red-and fatty beef meats with colorectal cancer risk. A slight increase in risk is detected for relatively high intake of cold cuts, sausages and bovine viscera, whereas lean beef is associated with a decreased risk [START_REF] Navarro | Characterization of meat consumption and risk of colorectal cancer in Córdoba, Argentina[END_REF].

Other investigations show that red meat intake is unrelated to colon cancer [START_REF] Lemarchand | Well-done red meat, metabolic phenotypes and colorectal cancer in Hawaii[END_REF][START_REF] Sinha | Meat, meat cooking methods and preservation, and risk for colorectal adenoma[END_REF] although individuals with low intake present lower risk for rectal cancer [START_REF] Lemarchand | Well-done red meat, metabolic phenotypes and colorectal cancer in Hawaii[END_REF]. Three wide studies were performed in Norway [START_REF] Gaard | Dietary factors and risk of colon cancer: a prospective study of 50,535 young Norwegian men and women[END_REF] and Sweden [START_REF] Augustsson | Dietary heterocyclic amines and cancer of the colon, rectum, bladder, and kidney: a population-based study[END_REF]Larsson et al. 1996). Although in these countries the HAA concentrations in the diet are usually lower than in other western European countries, differences are found when high consumption [START_REF] Augustsson | Dietary heterocyclic amines and cancer of the colon, rectum, bladder, and kidney: a population-based study[END_REF]Larsson et al. 1996) or very high frequency of consuming [START_REF] Gaard | Dietary factors and risk of colon cancer: a prospective study of 50,535 young Norwegian men and women[END_REF] are studied. So, high frequency of sausages consumption shows a relative risk of colon cancer of 3.5 (95% CI: 1.02-11.9) only for women. The RR for total meat consumption is 1.87. However, both parameters are not statistically significant for men [START_REF] Gaard | Dietary factors and risk of colon cancer: a prospective study of 50,535 young Norwegian men and women[END_REF]). In the prospective study for 61433 Swedish women, with ages between 40 and 75 years, a significant positive association between red meat consumption and risk of distal colon cancer is observed, but not of cancers of proximal colon nor rectum (Larsson et al. 1996). In a case-control study, risks for colon and rectal cancers are lower for the highest estimated intake category than for the lowest intake category of HAAs. Relative risks for each one of the HAAs are similar. Intake of meat and fish together, without considering the cooking method, is associated with increased risk of colon and bladder cancer [START_REF] Augustsson | Dietary heterocyclic amines and cancer of the colon, rectum, bladder, and kidney: a population-based study[END_REF].

Consumption of white meats is generally associated with lower cancer rates, even when these meats have HAAs contents greater than red meats (Sanz Alaejos and Afonso). Furthermore, chicken eaten without skin seems to decrease colorectal cancer risk [START_REF] Lemarchand | A case-control study of diet and colorectal cancer in a multiethnic population in Hawaii (United States): lipids and foods of animal origin[END_REF]). Red meat is no more implicated than any other meat type [START_REF] Kampman | Meat consumption, genetic susceptibility, and colon cancer risk: a United States multicenter casecontrol study[END_REF][START_REF] Lemarchand | Well-done red meat, metabolic phenotypes and colorectal cancer in Hawaii[END_REF][START_REF] Sinha | Dietary heterocyclic amines and the risk of lung cancer among Missouri women[END_REF][START_REF] Sinha | Meat, meat cooking methods and preservation, and risk for colorectal adenoma[END_REF].

Cooking methods:

The influence of cooking methods and cancer risk was also evaluated by the WCRF/AICR (1997). The report established that there is not convincing evidence that any cooking method modifies the risk of any cancer; neither there is evidence of any probably causal relationship. However, high intakes of grilled/barbecued red meat possibly increase the risk of stomach and colorectal cancer (WCRF/AICR, 1997). Higher risks are also observed for methods that require higher temperatures and close contact of the food to the heating source, such as barbecuing or iron-pan cooking and especially for red meats. Iron-pan means a kind of ground cast iron skillet used in Argentina to cook meat without adding oil or fat. No associations are found for boiled meats [START_REF] Navarro | Meat cooking habits and risk of colorectal cancer in Córdoba, Argentina[END_REF]. However, the results of studies on cooking methods are not consistent. Cooking methods hypothetically considered "hazardous", such as frying and broiling, were found to increase colorectal cancer risk in some studies but not in others [START_REF] Jägerstad | Genotoxicity of heat-processed foods[END_REF][START_REF] Lemarchand | Well-done red meat, metabolic phenotypes and colorectal cancer in Hawaii[END_REF][START_REF] Navarro | Meat cooking habits and risk of colorectal cancer in Córdoba, Argentina[END_REF][START_REF] Norat | Meat consumption and colorectal cancer: a review of epidemiologic evidence[END_REF]. The association between colorectal cancer and total HAAs intake is limited to men, and it is more clearly observed with MeIQx. This relation with MeIQx seems to result partially from an association with pan-fried meat and fish [START_REF] Lemarchand | Well-done red meat, metabolic phenotypes and colorectal cancer in Hawaii[END_REF]. Furthermore, MeIQx intake is associated with slightly increased risk of lung cancer.

However, DiMeIQx and PhIP intakes show no association [START_REF] Sinha | Dietary heterocyclic amines and the risk of lung cancer among Missouri women[END_REF]. When various factors are controlled, such as total-, red-, barbecued-or smoked meat, a statistically significant lung cancer risk associated to MeIQx remains. This MeIQx effect is still present, but no longer statistically significant, when the model includes well-done-or fried red meat. The opposite is also true; the previously significant effects of well-done and fried red meat are no longer significant after adjusting for MeIQx [START_REF] Terry | Dietary intake of heterocyclic amines and cancers of the oesophagus and gastric cardia[END_REF]. Grilling/barbecuing, oven broiling or intakes of well-done meat and fish do not appear to affect cancer risk [START_REF] Lemarchand | Well-done red meat, metabolic phenotypes and colorectal cancer in Hawaii[END_REF][START_REF] Sinha | Dietary heterocyclic amines and the risk of lung cancer among Missouri women[END_REF]. The exception appears when a darkly browned surface is preferred but excluding red roasted meat.

Roasted chicken and all barbecued meats were associated with a risk for colorectal cancer [START_REF] Navarro | Meat cooking habits and risk of colorectal cancer in Córdoba, Argentina[END_REF]). Pancreatic cancer cases reported high levels of grilled/barbecued meat intake and low levels of broiled meat intake relative to controls. Other meat variables do not show statistically significant associations with risk, neither alter the findings for barbecued meat intake.

These meat variables include processed-, total-, total broiled-, total fried-, total red-, or total meat cooked by means other than grilling [START_REF] Anderson | Meat intake and cooking techniques: associations with pancreatic cancer[END_REF]. In a population-based case-control study [START_REF] Anderson | Dietary intake of heterocyclic amines and benzo(a)pyrene: Associations with pancreatic cancer[END_REF], dietary intake of HAAs and benzo(a)pyrene formed in grilled/barbecued red meat and fried red meat, was studied in relation to pancreatic cancer. Cases, when compared with controls, had higher mean levels of all the carcinogens studied (PhIP, MeIQx, DiMeIQx, and benzo(a)pyrene), as well as total mutagenic activity. The highest ORs were seen in the highest quintiles of intake and, with the exception of MeIQx, there were all statistically significant. Grilled/barbecued red meat intake was a statistically significant predictor of pancreatic cancer risk. Fried red meat intake also increased risk, but was not statistically significant, because fried meats in general do not contain appreciable concentrations of PAHs [START_REF] Anderson | Dietary intake of heterocyclic amines and benzo(a)pyrene: Associations with pancreatic cancer[END_REF].

3. Doneness: Meat doneness was weakly and inconsistently associated with prostate cancer risk for individual types of meat, but an increased risk was observed for very well-done beefsteak [START_REF] Norrish | Heterocyclic amine content of cooked meat and risk of prostate cancer[END_REF]). The incidence rates of prostate, pancreas and colon cancers seem to be positively related to exposure to HAAs among men of different races in Los Angeles County (USA).

This result can partially explain the higher incidence among blacks with respect to white men in the studied human group, because black men consume higher amounts of well-done meats, bacon, pork and cold cuts [START_REF] Bogen | US dietary exposures to heterocyclic amines[END_REF]. Moreover, an increased risk of breast cancer is observed in healthy women who consume very well-done meats. PhIP is detected in their milks at levels of 59 pg/mL, but not in milk from a vegetarian donor. Preference for well-done red meat is not significantly associated with colon cancer, but seems to increase risk somewhat for rectal cancer [START_REF] Lemarchand | Well-done red meat, metabolic phenotypes and colorectal cancer in Hawaii[END_REF]Sinha et al. 1999). On the other hand, MeIQx, PhIP, and benzo{a}pyrene were associated with a slight increased risk of adenoma of colon, non-advanced adenoma, and single adenomas in a recent and wide population-based case-control study, related to a high intake of well-done red meat, bacon and sausage. However, in a fully adjusted model, total mutagenicity, DiMeIQx, and MeIQx intakes are not associated with colorectal adenoma risk when all adenomas were combined. Rectal cancer is not associated with any of the meat variables, such as meat types or doneness/cooking methods [START_REF] Sinha | Meat, meat cooking methods and preservation, and risk for colorectal adenoma[END_REF]. Pancreatic cancer cases reported slightly higher mean levels of intake for total-, total red-, and processed meat, and lower mean levels of intake for white meat, when comparing with controls. The relative amounts of meat intake by doneness levels are not different between cases and controls [START_REF] Anderson | Meat intake and cooking techniques: associations with pancreatic cancer[END_REF]. However, other studies have shown no such association among well-done red meat consumption and cancer [START_REF] Augustsson | Dietary heterocyclic amines and cancer of the colon, rectum, bladder, and kidney: a population-based study[END_REF][START_REF] Gunter | Meat intake, cooking-related mutagens and risk of colorectal adenoma in a sigmoidoscopy-based casecontrol study[END_REF][START_REF] Kampman | Meat consumption, genetic susceptibility, and colon cancer risk: a United States multicenter casecontrol study[END_REF][START_REF] Norrish | Heterocyclic amine content of cooked meat and risk of prostate cancer[END_REF][START_REF] Sinha | Dietary heterocyclic amines and the risk of lung cancer among Missouri women[END_REF][START_REF] Sinha | Dietary intake of heterocyclic amines, meat-derived mutagenic activity, and the risk of colorectal adenomas[END_REF].

As conclusion, in general, the three high-temperature cooking methods (pan-frying, ovenbroiling and grilling/barbecuing) seem to produce the highest HAAs concentrations. Preparation methods like boiling, oven roasting, deep-frying, and charcoal grilling are generally "milder", and However, there is insufficient scientific evidence to prove that these genotoxic compounds really cause human cancer, and so, no limits have been set for their presence in cooked foods.

Nevertheless, the competent authorities in most Western countries recommend minimizing their occurrence. The reasons are the co-existence of other mutagens/carcinogens in foods, and the possibility that the HAAs induce mutations, therefore increasing the sensitivity to tumor promoters.

So, some recommendations could be followed: Choose lean cuts. Apply lower temperatures and shorter cooking times. Avoid browning of foods or, at least, remove the crust and charred parts of fried of grilled meat, poultry and fish. Do not allow drippings from meat to become dry before making gravy. To lessen the levels of HAAs produced it may be useful cooking meat and fish together with foodstuffs containing phenolic antioxidants or with tomatoes, carrots or other vegetables rich of carotenoids and antioxidant vitamins.

Uncertainties associated to epidemiological results

The lack of consistency of the results for all types of meats together with the lack of information from cohort studies do not always permit a judgment about the possible differences in the effect of meat on e.g., colon and rectal cancer risks, separately [START_REF] Norat | Meat consumption and colorectal cancer: a review of epidemiologic evidence[END_REF]. In any case, the present data are not enough to evaluate the dose response of the possible relationship between HAAs and human cancer, in its widest sense. Besides, a simplistic approach to evaluate only HAAs with respect to cancer risk may not be too appropriate, taking into account that diet is a complex mixture that contains carcinogens, co-carcinogens and anti-carcinogens [START_REF] Jägerstad | Genotoxicity of heat-processed foods[END_REF]; Urinary excretion of MeIQx and its metabolites has been also studied [START_REF] Ji | Urinary excretion of 2-amino-3,8-dimethyl-imidazo[4,5-f]-quinoxaline in white, black, and Asian men in Los Angeles County[END_REF][START_REF] Lynch | Intra-and interindividual variability in systemic exposure in humans to 2-amino-3,8-dimethyl-imidazo[4,5f]quinoxaline and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, carcinogens present in cooked beef[END_REF][START_REF] Sinha | Lower levels of urinary 2-amino-3,8-dimethylimidazo[4,5-f]-quinoxaline (MeIQx) in humans with higher CYP1A2 activity[END_REF][START_REF] Stillwell | Human urinary excretion of sulfamate and glucuronide conjugates of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx)[END_REF]Stillwell et al. , 1999aStillwell et al. , 1999b;;[START_REF] Turesky | Metabolism of the food-borne mutagen 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) in humans[END_REF]. MeIQx metabolism and disposition in humans are more strongly influenced by CYP1A2 activity than those of PhIP. Biotransformation of MeIQx via CYP1A2 oxidation to form 8-CH 2 OH-MeIQx and the hydroxylamine NHOH-MeIQx, followed by N-2-glucuronidation is a general pathway for the MeIQx metabolism in humans. The formation of both 8-CH 2 OH-MeIQx and NHOH-MeIQx is catalyzed by CYP1A2 and may be useful biomarkers of CYP1A2 activity in humans [START_REF] Turesky | Metabolism of the food-borne mutagen 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) in humans[END_REF]. Low levels of total unconjugated MeIQx are associated with high CYP1A2 activity when adjusted for amount of ingested meat, whereas NAT2 activity shows no relationship with the latter [START_REF] Sinha | Lower levels of urinary 2-amino-3,8-dimethylimidazo[4,5-f]-quinoxaline (MeIQx) in humans with higher CYP1A2 activity[END_REF]. However, significant interracial differences are observed for black, white and Asian men. This is because urinary levels of MeIQx are positively associated with intake frequencies of pork, bacon and cold cuts [START_REF] Ji | Urinary excretion of 2-amino-3,8-dimethyl-imidazo[4,5-f]-quinoxaline in white, black, and Asian men in Los Angeles County[END_REF].

The theoretical cancer risk by HAAs in the Swiss diet is calculated by two different procedures and results in 0.7•10 -5 or, roughly, 10 -5 (Zimmerli et al. 2001). If it is assumed that humans may be 10 to 100 times more susceptible to HAAs than male rats, the hypothetical excess cancer risk would be in the order of 10 -4 to 10 -3

. Cancer potency estimates for several of the HAAs were as high as one in a 100,000 per ppb in the total diet. Based on HAAs exposures ranges of 0.02 to 83 ppb in the human total diet, and rodent data, [START_REF] Gaylor | Quantitative cancer risk assessment of heterocyclic amines in cooked foods[END_REF] [START_REF] Bogen | Cancer potencies of heterocyclic amines found in cooked foods[END_REF][START_REF] Felton | Chemical analysis, prevention, and low-level dosimetry of heterocyclic amines from cooked food[END_REF][START_REF] Gaylor | Quantitative cancer risk assessment of heterocyclic amines in cooked foods[END_REF][START_REF] Layton | Cancer risk of heterocyclic amines in cooked foods. An analysis and implications for research[END_REF]. [START_REF] Adamson | Extrapolation of heterocyclic amine carcinogenesis data from rodents and nonhuman primates to humans[END_REF] suggests potential risks of 10 -4 to 10 -3

. On the other hand, interspecies differences in CYP1A2 enzyme expression and catalytic activities may be significant and must be carefully considered when assessing human health risk [START_REF] Kim | Cytochrome P450 activation of arylamines and heterocyclic amines[END_REF].

So, important differences between human and rat CYP1A2 were found in the C8-and N-oxidation of MeIQx [START_REF] Turesky | Regioselective differences in C8 and N-oxidation of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline by human and rat liver microsomes and cytochromes P450 1A2[END_REF], and in the levels of expression and catalytic activities for HAAs Nhydroxylation [START_REF] Turesky | Interspecies differences in metabolism of heterocyclic amines by rat and human P450 1A2[END_REF], although rat and human CYP1A2 have identical 75% amino acid sequence [START_REF] Guengerich | Comparison of catalytic selectivity of cytochrome P450 subfamily members from different species[END_REF]. The value 10 -3 is considered as a maximum credible risk [START_REF] Felton | Chemical analysis, prevention, and low-level dosimetry of heterocyclic amines from cooked food[END_REF]. Similar theoretical cancer risks result from human exposure to other compounds unavoidably present in the diet, such as naturally occurring radionuclides (potassium 40) or excess calories (Zimmerli et al. 2001).

There are several major uncertainties in the present estimation of cancer risk, including human susceptibility, exposure, carcinogenic potencies of individual HAA, as well as possible interactions between different HAAs and other substances normally present in a diversified diet.

The most likely target organs for most HAAs are the liver and the gastrointestinal tract.

Nevertheless, in Switzerland the cancer mortality trends for these organs (gastric and colon cancers) show a permanent slight decrease, although meat, fish and alcohol consumption have greatly increased in Switzerland over the last 50 years, and milk intake (including yogurt) has decreased (Zimmerli et al. 2001).

A major trouble of the epidemiological studies is the obtaining of a correct estimation for the exposure to HAAs [START_REF] Alexander | Biomarkers of exposure to heterocyclic amines: approaches to improve the exposure assessment[END_REF][START_REF] Keating | Methods for estimating heterocyclic amine concentrations in cooked meats in the US diet[END_REF][START_REF] Keating | Factors determining dietary intakes of heterocyclic amines in cooked foods[END_REF][START_REF] Sinha | Exposure assessment of heterocyclic amines (HCAs) in epidemiologic studies[END_REF][START_REF] Skog | Problems associated with the determination of heterocyclic amines in cooked foods and human exposure[END_REF]. Dietary assessment in combination with analytical data on HAAs levels and variety of food samples is not an easy task. Consequently, it is not surprising that bibliographic data are very incomplete (Sanz Alaejos and Afonso). Table IV shows some estimates for HAAs mean intakes in various countries. The dispersion of the results is evident, and there are scarce data referred to individual HAAs.

Insert Table IV about here Some attempts have been made to develop biomarkers in order to overcome some of the problems associated with the use of questionnaires in the assessment of HAAs exposure.

Biomarkers of diet promise to provide a more accurate measure of dietary intake. They are more objective because they do not rely on the subject's memory when being interviewed, or on the accuracy of recording the food diary. The use of biomarkers does not solve the problem of exposure estimation back in time for years, since HAAs do not accumulate in body tissues. Hence, HAAs in body fluids or tissues would only give a measure of recent exposure [START_REF] Frandsen | 2-Amino-1-methyl-6-(5-hydroxy-)phenylimidazo[4,5b]pyridine (5-OH-PhIP), a biomarker for the genotoxic dose of the heterocyclic amine 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)[END_REF][START_REF] Lynch | Intra-and interindividual variability in systemic exposure in humans to 2-amino-3,8-dimethyl-imidazo[4,5f]quinoxaline and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, carcinogens present in cooked beef[END_REF][START_REF] Stillwell | Urinary excretion of unmetabolized and phase II conjugates of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline in humans: relationship to cytochrome P4501A2 and N-acetyltransferase activity[END_REF][START_REF] Strickland | Measurement of 2-amino-1-methyl-6-phenylimidazo[,5b]pyridine (PhIP) in acid-hydrolyzed urine by high-performance liquid chromatography with fluorescence detection[END_REF][START_REF] Wild | A critical evaluation of the application of biomarkers in epidemiological studies on diet and health[END_REF]). However, these short-term markers are useful tools to study mechanisms of action of HAAs, and to validate questionnaires [START_REF] Sinha | An epidemiologic approach to studying heterocyclic amines[END_REF]. So, Peters et al. ( 2003) when combining the measurements of urinary mutagenicity and the estimated intake of meat-derived mutagenicity, found a stronger positive association with risk for colorectal adenomas than did either variable separately, when comparing the highest with the lowest quintile of combined mutagenicity (OR=5.6; 95% CI=2.3-13.9). In consequence, urinary mutagenicity may be considered a useful adjunct to questionnaire data and may have value as a direct biomarker of exposure and/or risk for colorectal adenoma (Peters et al. ). Also, the formation of both 8-CH 2 OH-MeIQx and NHOH-MeIQx may be useful biomarkers of CYP1A2 activity in humans [START_REF] Turesky | Metabolism of the food-borne mutagen 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) in humans[END_REF]).

On the other hand, protein adducts in blood and HAAs in hair are biomarkers reflecting longer-term exposure. The use of PhIP (and other HAAs) in hair is attractive. It would integrate the dose over several months depending on the length of the hair, which grows approximately 1 cm per month. Hair is easy to sample and store for the purpose of later analysis in prospective studies on the relationship between HAAs exposure and cancer [START_REF] Alexander | Biomarkers of exposure to heterocyclic amines: approaches to improve the exposure assessment[END_REF][START_REF] Kobayashi | 2-Amino-1-methyl-6-phenylimidazo[4,5b]pyridine (PhIP) level in human hair as biomarker for dietary grilled/stir-fried meat and fish intake[END_REF].

A main problem when using literature data related to HAAs contained in cooked dishes is that many experiments have been performed under unspecified cooking conditions, or using high cooking temperatures during a long time to maximize the HAAs production. This last case can lead to a non-representative form of the usual way of cooking meats by the general population in a certain country. The relation between the degree of doneness and the surface browning may differ because some people fry their meats at a high temperature for a short time to get a brown surface but the interior is not so cooked through, while others fry their meats at low temperatures but during a longer time. This can lead to the same degree of surface browning but very different HAAs amounts. The colored photos are sometimes used to assess the preference of the consumer on the degree of doneness and, indirectly, the amount of HAAs [START_REF] Alexander | Biomarkers of exposure to heterocyclic amines: approaches to improve the exposure assessment[END_REF][START_REF] Keating | Methods for estimating heterocyclic amine concentrations in cooked meats in the US diet[END_REF][START_REF] Keating | Factors determining dietary intakes of heterocyclic amines in cooked foods[END_REF][START_REF] Sinha | Exposure assessment of heterocyclic amines (HCAs) in epidemiologic studies[END_REF][START_REF] Skog | Problems associated with the determination of heterocyclic amines in cooked foods and human exposure[END_REF]. The results seem to lead to an underestimation of exposure [START_REF] Skog | Problems associated with the determination of heterocyclic amines in cooked foods and human exposure[END_REF]. However, HAAs levels in home-cooked meat samples are significantly different when samples are visually classified for doneness, but not when selfreported doneness preference is used to classify doneness or to categorize samples [START_REF] Keating | Comparison of heterocyclic amines levels in home-cooked meats with exposure indicators (United States)[END_REF]. HAAs levels in meats obtained from homes vary considerably from the laboratory data. In a study [START_REF] Knize | Factors affecting human heterocyclic amine intake and the metabolism of PhIP[END_REF], samples were taken from volunteers responding in survey that they preferred their meat well-done or very well-done, leading to expect high HAAs exposures in these households. But, surprisingly, in that collection of meats samples, approximately 25% of the samples had undetectable amounts of MeIQx or PhIP. Besides, although well-done chicken breast prepared in the laboratory may contain large amounts of PhIP, a survey of flame-grilled meat samples cooked in private homes showed PhIP levels in beefsteak and chicken breast that were not significantly different. The extremely high PhIP levels reported in some studies of grilled chicken are not observed in home-cooked samples [START_REF] Knize | Factors affecting human heterocyclic amine intake and the metabolism of PhIP[END_REF]. Considerable differences between equivalent products from different manufacturers are found in many cases. In addition, the terminology used for different cooking methods varies around the world. In Argentina and Uruguay the term "roasting/broiling" does not expose foods directly to flame, whereas "grilling" may use a hot surface (i.e., a pre-heated heavy iron plate), or a grill directly above burning coals [START_REF] Matos | Review on meat consumption and cancer in South America[END_REF][START_REF] Navarro | Meat cooking habits and risk of colorectal cancer in Córdoba, Argentina[END_REF]). Cooking practices and eating habits differ between different populations, and conclusions drawn from one population may not be applicable to another [START_REF] Alexander | Biomarkers of exposure to heterocyclic amines: approaches to improve the exposure assessment[END_REF][START_REF] Ferguson | Meat consumption, cancer risk and population groups within New Zealand[END_REF]Keating andBogen 2001, 2004;[START_REF] Keating | Factors determining dietary intakes of heterocyclic amines in cooked foods[END_REF][START_REF] Matos | Review on meat consumption and cancer in South America[END_REF][START_REF] Sinha | Exposure assessment of heterocyclic amines (HCAs) in epidemiologic studies[END_REF][START_REF] Skog | Problems associated with the determination of heterocyclic amines in cooked foods and human exposure[END_REF]. In some countries pan residues are used for preparing gravies, and pan residues contain substantial amounts of HAAs compared with the corresponding food item. Besides, meat extracts and bouillon (cubes) contribute to the HAAs intake. Therefore, it is important to establish databases on HAAs content in cooked foods that are representative for the eating habits of the population being studied, and taking into account each ingredient of the recipe, spices, condiments, etc. The dishes should be prepared in a way that reflects regular household and restaurant cooking conditions.

Really, recent advances in the analytical instrumentation, concretely in LC-MS and GC-MS, have greatly facilitated the ability to measure HAAs in foods, as well as HAAs and their metabolites in urine of meat eaters. Nevertheless, the accurate determination of HAAs is a difficult analytical task since traces of these compounds have to be determined in highly complex food matrices. This problem can only be solved by combining both elaborate sample preparation steps with selective separation steps, and then followed by sensitive detection methods to quantify low levels of HAAs. Pyridoindole derivatives:

N N N N NH 2 CH 3 2-amino-3,4-dimethyl-imidazo(4,5-f)-quinoxaline 4-MeIQx N N N N NH 2 CH 3 CH 3 2-amino-3,8-dimethyl-imidazo(4,5-f)-quinoxaline 8-MeIQx N N N N NH 2 C H 3 CH 3 2-amino-3,7,8-trimethyl-imidazo(4,5-f)-quinoxaline 7,8-DiMeIQx N N N N NH 2 C H 3 C H 3 CH 3 2-amino-3,4,8-trimethyl-imidazo(4,5-f)-quinoxaline 4,8-DiMeIQx N N N N NH 2 CH 3 C H 3 CH 3 2-amino-4-hydroxymethyl-3,8-dimethyl-imidazo(4,5-f)- quinoxaline 4-CH2OH-8- MeIQx N N N N NH 2 C H 3 CH 2 OH CH 3 2-amino-3,4,7,8-tetramethyl-imidazo(4,5-f)-quinoxaline TriMeIQx N N N N NH 2 C H 3 C H 3 CH 3 CH 3 2-amino-1,7,9-trimethyl-imidazo(4,5-g)-quinoxaline 7,9-DiMeIgQx N N N N NH 2 CH 3 C H 3 CH 3 F o r P e e r
α -carbolines 2-amino-9H-pyrido(2,3-b)indole AαC N N NH 2 H 2-amino-3-methyl-9H-pyrido(2,3-b)indole MeAαC N N NH 2 CH 3 H β -carbolines 1-methyl-9H-pyrido(3,4-b)indole Harman N N CH 3 H 9H-pyrido(3,4-b)indole Nor-harman N N H γ -carbolines 3-amino-1-methyl-5H-pyrido(4,3-b)indole Trp-P-2 N N NH 2 C H 3 H 3-amino-1,4-dimethyl-5H-pyrido(4,3-b)-indole Trp-P-1 N NH 2 C H 3 N CH 3 H F o r P e e r

  of HAAs are formed. The degree of doneness, which is often closely related to surface browning and total cooking time, is a key issue for HAAs production in cooked meat. The formation of the crust is the result of steady transportation of water and dissolved compounds such as amino acids and creatinine to the surface by capillary flow to the evaporation zone. Thus, the precursors of HAAs are concentrated on or near the surface of the meat where the temperature is highest.

  the development of cancer takes a long time, and so there is a delay of several years between exposure and disease outcome.Metabolic aspects of HAAs have been reviewed[START_REF] Gooderham | Food-derived heterocyclic amine mutagens: Variable metabolism and significance to humans[END_REF] Sanz Alaejos et al. b). The major pathway for the metabolic activation of HAAs starts with the N-hydroxylation of the exocyclic amino group, catalyzed mainly by cytochrome P450 1A2 (CYP1A2). Although these metabolites may directly react with DNA, this N-hydroxylation step is usually followed by sulphation or acetylation by means of sulfotransferase 1A1 (SULT1A1), or N-acetyltransferases (NAT), respectively(Sanz Alaejos et al. b).

  must be used. In this sense, food frequency questionnaires including not only the type and amount of foods consumed but also the cooking methods and the level of doneness are required. There are several flaws including bias, inconsistent reporting, difficulty in quantifying cooking doneness by such methods, and the day-to-day variation in the diet. Moreover, HAAs belong to a class of numerous compounds and the quantification of each of them in a large number

  Tedious clean-up procedures that include extraction, purification, and pre-concentration steps, followed by a separation technique, such as liquid or gas chromatography and capillary electrophoresis are usually used. The main detection systems used are UV, fluorescence, electrochemical and MS(Sanz Alaejos et al. a). In addition, the different analytical methods applied comparable. Some problems in determining appropriate estimates of extraction recovery rates must also be taken into account. Some HAAs or HAAs derivatives can bind with other food components. All of these formed compounds cannot be extracted from food by the usual extraction methods and, therefore, the dietary assessment of genotoxic compounds may be underestimated. Therefore, different extraction procedures have to be applied to cooked and uncooked meat, before and after enzymatic proteolysis. Consequently, the use of absolute or relative values of HAAs content in food can lead to erroneous conclusions when possible cancer risks are established for the intake of these foods.Divergent results can be due to several causes, one of them is the meat type and the ingredients added. Also, analytical results on HAAs content in foods obtained under unspecified cooking conditions or at high cooking temperatures may be not representative. It should very convenient: a) to establish databases on HAAs content in cooked foods that are representative for the eating habits of the population being studied, and taking into account each ingredient of the recipe; b) to record the inside and outside food appearance in the food frequency questionnaires, instead of simply recording the "doneness level"; c) to consider the possible role of HAAs in the cancer development in conjunction with PAHs and other toxic compounds; and d) to use biomarkers in order to determine the HAAs exposure.

F

ISOLATED

  on cancer risk due to HAAs formed during cooking is on the order of one per 10,000.Although rodents, monkeys and humans have the capacity to activate HAAs, studies using hepatic microsomes demonstrated that humans have a greater capacity to activate HAAs than rodents or cynomolgus monkeys. Human risk, based on linear extrapolation of TD 50 calculations from mouse, rat, or primate tumor data, and on mean estimated mutagen exposures for the U.S. population,

	13
	o F the upper limit suggests potential risks of 10 -5 to 10 -3
	r
	P
	e e r
	R e v i e w
	O n
	l y
	estimated that

Table II .

 II Influence of HAAs consumption on cancer risk development

	Page 35 of 52 Page 37 of 52 Page 39 of 52 Page 43 of 52					Food Additives and Contaminants Food Additives and Contaminants Food Additives and Contaminants Food Additives and Contaminants Food Additives and Contaminants Food Additives and Contaminants Food Additives and Contaminants Food Additives and Contaminants Food Additives and Contaminants Food Additives and Contaminants		Page 34 of 52 Page 36 of 52 Page 40 of 52
									31 32 33 34 35 36 37 38 39 40
	Esophagus (squamous cell) Pancreas (exocrine)		273 513 145 150 256 193 688	657 513 200 745 284 674 34817	1.54 0.8 1.74 1.04 2.11 2.4 1.5 Non significant 0.96-2.47 0.5-1.2 1.23-2.45 0.90-1.19 0.90-4.92 1.2-4.8 Significant 0.9-2.7	Very well-done hamburger Grilled/barbecued meat and fish Fried red meat, darkly browned Processed red meat with N-nitroso compounds Very well-done red meat Fried meats, pan juices Red meat, fried meat MeIQx; well-done barbecued meat and pan-fried meat PhIP, MeIQx, DiMeIQx, benzo{a}pyrene	USA (Iowa) USA (Hawaii) LeMarchand et Zheng et al. Sweden Terry et al. 2003 Uruguay Deneo Pellegrini USA Anderson et al. USA Sinha et al. 2005
	Esophagus (adenocarcinoma)	64 157 952	198 1205	1.64 0.9 1.51 1.05 1.15 0.7 1.8 1.33	0.92-2.93 0.6-1.3 0.98-2.32 0.92-1.22 0.97-1.36 0.4-1.2 1.0-3.1 0.98-1.79	Very well-done bacon Well-done meat and fish All meats and methods, lightly browned Total meat Pan-fried red meat High intake of MeIQx, 4,8-DiMeIQx, and PhIP PhIP; well-done barbecued meat and pan-fried meat Well-done red meat (men)	USA (California	1998 al. 2002 et al. 1996 2005 Murtaugh et al.
	Cancer sites Aero-digestive Bladder Colon Colorectal Colorectal adenomas Gastric cardia (adenocarcinoma) Kidney Lung Pancreas Prostate Rectum Stomach	Cases Controls Odds ratios Non significant 95% confidence interval 2.0 1.0-4.3 2.2 1.1-4.2 140 286 2.8 1.4-6.0 0.8 0.5-1.5 1.2 0.7-2.1 273 553 5.0 3.3-7.6 Non significant 0.38 0.23-0.64 144 576 4.04 2.24-7.27 F o r P e e r R e v i e w Intakes (observations) Fried meat, white meat Broiled meat Total HAAs Red meat Total meat and fish HAAs or PhIP (Current smoking) Well-done fried meat Fruits, cooked vegetables, potatoes, cheese Barbecued meat, salted meat, fried eggs, N-nitroso compounds O n l y 2.21 1.30-3.77 Very well-done beef steaks 4.64 1.36-15.70 Very well-done meats 0.6 0.4-1.0 HAAs 0.9 0.5-1.4 Total meat and fish 352 553 1.0 0.7-1.4 (current smoking) 0.9 Red and white meat 1.2 0.9-1.5 Well-done red meat 1542 1860 1.4 1.0-1.9 Processed meat (men) 1.11 0.97-1.27 DiMeIQx; red meat (processed, roasted) 1.17 1.01-1.35 PhIP; red meat (processed, roasted) 1.18 1.01-1.38 MeIQx; red meat (processed, roasted) 2474 34817 1.18 1.02-1.35 Benzo{a}pyrene; red meat (processed, roasted) F o r P e e r R e v i e w O n l y 1.0 0.7-1.5 Total red meat; red meat, well-or very well-done 1.3 0.8-1.9 Pan-fried/oven broiled meat and fish 1.0 0.6-1.1 MeIQx (men) 1.0 0.6-1.6 PhIP (men) 289 289 1.1 0.7-1.7 4,8-DiMeIQx (men) 1.7 1.2-2.5 Well-, very well-done red meat 1.8-2.0 4,8-DiMeIQx 620 1038 2.0 1.3-3.2 Red meat, pan-fried red meat 88 200 0.74 0.52-1.05 Boiling/stewing white meat, >21.6 g/day 87 200 0.93 0.65-1.34 Boiling/stewing red meat, >31.2 g/day 110 1.08 0.76-1.54 Roasted red meat, darkly browned 148 2.19 1.53-3.12 Roasted white meat, darkly browned 199 F o r P e e r R e v i e w O n l y 189 202 4.57 3.10-6.73 All meats and methods, darkly browned 133 199 1.91 1.33-2.74 Barbecued white meat, darkly browned 176 200 2.85 1.97-4.10 Barbecued red meat, darkly browned 167 200 2.44 1.71-3.47 Iron-pan cooked red meat, darkly browned 0.5 0.2-1.07 Poultry and fish (women) 102 537 2.7 1.1-6.7 Red meat (men) 431 433 1.2 0.8-1.9 Meat 1.51 1.03-2.23 Total meat 500 500 1.97 1.30-2.98 Red meat 0.92 0.78-1.09 Broiled red meat 0.95 0.74-1.22 Pan-fried red meat 3696 34817 F o r P e e r R e v i e w O n l y 1.07 0.92-1.24 Red meat 1.14 1.00-1.30 High intake of bacon and sausages 1.21 1.06-1.37 Well-done red meat, as hamburgers, hot-dogs, sausages, bacon (HAAs+PAHs) 32 100 1.75 0.78-4.05 MeIQx low exposure 53 79 4.09 1.94-9.08 MeIQx high exposure 54 80 4.36 2.08-9.60 Well-, very well-done red meat 0.8 0.4-1.4 Total HAAs (women) 1.8 1.1-3.1 Total HAAs (men) 1.04 0.98-1.09 Total meat 0.19 0.01-2.86 Microwaved red meat 0.94 0.74-1.20 Broiled red meat 146 228 F o r P e e r R e v i e w O n l y 1.22 0.92-1.61 Well-, very well-done pan-fried red meat 1.26 1.06-1.50 Grilled/barbecued red meat 1.85 1.24-2.75 Well-, very well-done grilled/barbecued red meat 1.11 1.03-1.19 Red meat 146 228 1.29 1.08-1.54 Well-, very well-done red meat 2.1 1.0-4.3 MeIQx 2.2 1.2-4.1 4,8-DiMeIQx 2.5 1.1-5.5 PhIP 146 228 3.1 1.4-6.8 HAAs 488 488 2.2 1.1-4.3 Fried red meat, darkly browned (high frequent intake) 565 565 Non significant Well-done red meat (HAAs) F o r P e e r R e v i e w O n l y 233 1.3 0.8-2.1 1.0 0.5-1.9 HAAs or total meat and fish 138 553 1.3 0.8-2.1 (Current smoking) 0.78 0.45-1.39 Processed meat 2.07 1.03-4.19 High intake of barbecued meat 2.18 1.14-4.19 PhIP (>15.6 ng/d) 121 243 3.42 1.76-6.65 High intake of red meat 1.0 0.5-2.4 Stir-fried meat less often than daily (smokers, women) 2.7 1.3-5.5 Stir-fried meat daily (smokers, women) 3.7 1.8-7.5 Stir-fried meat daily (kitchen fined with oily fumes during cooking, women) 303 765 F o r P e e r R e v i e w O n l y 1.5 1.1-2.1 Fried or well-done red meat (women) 1.6 1.1-2.4 Total meat (women) 593 623 1.8 1.2-2.7 Red meat (women) 1.3 0.9-1.9 Fried or well-done red meat (women) 1.4 1.0-1.9 Total meat or red meat (women) 1.5 1.1-2.0 Barbecued red meat or smoked meat (women) 0.9 0.8-1.1 PhIP (women) 1.2 0.9-1.6 DiMeIQx (women) 593 623 1.5 1.1-2.0 MeIQx (women) 343 200 1.0 0.7-1.5 MeIQx (women, heavy smokers) 203 340 2.1 1.3-3.3 MeIQx (women, light/moderate smokers) 47 83 3.6 1.3-10.3 MeIQx (women, non smokers) F o r P e e r R e v i e w O n l y 2.0 1.2-3.5 DiMeIQx; well-done barbecued meat and pan-fried meat 2.2 1.2-4.0 Benzo{a}pyrene; well-done barbecued meat and pan-fried meat 2.4 1.3-4.3 Mutagenic activity; well-done barbecued meat and pan-fried meat 0.7 0.4-1.2 Broiled red meat 1.4 0.7-2.6 Fried red meat 193 674 2.2 1.4-3.4 Grilled/barbecued red meat Non significant Meat doneness and major HAAs Weakly significant IFP 317 480 1.68 1.02-2.77 Very well-done beef steaks 464 459 1.68 1.20-2.36 Well-done meat 0.7 0.4-1.1 HAAs 249 553 F o r P e e r R e v i e w O n l y 1.37 0.98-1.92 Mutagen index (men) 0.8 0.4-1.6 Oven broiled, grilled/barbecued meat and fish 1.2 0.7-2.0 Well-done meat and fish 1.5 0.9-2.4 Well-, very well-done red meat 1.6 1.0-2.5 Pan-fried meat and fish 210 210 1.7 1.0-3.0 Total red meat 1.7 0.3-3.8 PhIP (men) 2.7 1.1-6.3 4,8-DiMeIQx (men) 137 137 3.1 1.3-7.7 MeIQx (men) 1.7 0.7-4.0 Total meat and proteins, total energy and total fat 1.9 1.1-3.5 Energy adjusted for high intake of processed meat 123 282 F o r P e e r R e v i e w O n l y	Geographical location Uruguay Sweden Uruguay Sweden USA USA USA (North Caroline) Argentina (Córdoba) Germany Germany United Kingdom Barrett et al. Ref. De Stefani et al. 1998 Augustsson et al. 1999 Balbi et al. 2001 Augustsson et al. 1999 Kampman et al. 1999 Sinha et al. 2005 Butler et al. 2003 Navarro et al. 2004 Tiemersma et al. 2002 Tiemersma et al. 2004 2003 USA Sinha et al. 2005 USA (Arkansas and Tennessee) Nowell et al. 2002 USA (Hawaii) LeMarchand et al. 2002 USA (Maryland, Missouri) Sinha et al. 1999, 2001 USA (Missouri) Sinha et al. 2001 USA (California) Probsttensch et al. 1997 USA (California, Gunter et al. Sweden Augustsson et al. 1999 Uruguay De Stefani et al. 1998 China Seow et al. 2000 USA (Missouri) Sinha et al. 1998 USA (upper Midwest area) Anderson et al. 2002 New Zealand Norrish et al. 1999 USA Nowell et al. 2004 and Utah) 2004 USA (Hawaii) LeMarchand et al. 2002 Uruguay De Stefani et al. 2001
	Breast Lung, squamous cell carcinoma Lung, other histological types	352 128	382	3.34 5.3 1.42 0.99 1.01 4.3 1.9 1.0 4.6 1.03 1.10 5.0 1.6 0.9 Non significant 1.85-6.02 2.8-10.2 1.00-2.02 0.80-1.23 0.79-1.28 2.7-6.8 1.2-3.1 0.6-1.6 2.3-9.0 0.91-1.16 0.96-1.26 3.4-7.3 1.1-2.5 0.6-1.4	For IQ, in red meat, total meat, beef, fried meat (OR: 2.6 for PhIP) Grilled meat Baked red meat Fumes from meat cooking (ex-smokers, women) MeIQx (women) Total meat and fish Total meat Fried white meat, darkly browned Fried red meat White meat Rare-, medium-done red meat Fumes from meat cooking (current smokers, women) MeIQx (women) (Current smoking) Fried, barbecued, salted meat and PhIP	Uruguay southern)	De Stefani et al. 2005 1997

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Table III .

 III Influence of foods/nutrients intake on cancer risk development

	Page 45 of 52		Food Additives and Contaminants Food Additives and Contaminants Food Additives and Contaminants		Page 46 of 52
						42 43
		For distal colon: total meat (men: 2.20) Vegetables and fruits (women: 0.48), Red meat (1.54); refined grain (1.32); Vegetables, fruit, garlic, whole grain,	Total meat (women: 0.94; men: 1.17),	USA (Seattle) Switzerland	Shannon et al. Levi et al. 1999
		pork or processed meat (1.27)	cereals (women: 0.47; men: 0.43), dairy coffee	red meat (women: 0.72; men: 1.48)	(Canton of Vaud)	1996
	Cancer sites Bladder Brain Breast Colon Colorectal	Higher risk (odds ratio) Red meat (1.6) Salted vegetables, salted fish Well-done deep-fried red meat (1.92), freshwater fish Red meat (1.2) High intake of preserved animal or vegetal foods (men: 2.0; women: 2.7), eggs (1.3), fish (men: 1.7), red meat F o r P e e r R e v i e w Protective effect (odds ratio) No association (odds ratio) Vegetables, fruit, fresh fish, poultry, Ca, vitamins E, C, β-carotene Non-hydrogenated soybean oil (0.48) Fresh fruit (0.6), vitamins E, C, β-carotene, fiber Fish (women: 1.2) O n l y Italy (Northern) Geographical location China (Northeast) Hu et al. 1999 Ref. Tavani et al. 2000 China (Shanghai) Dai et al. 2002 Italy (Northern) Tavani et al. 2000 China (Shanghai) Chiu et al. 2003 Red meat (1.9) Italy (Northern) Tavani et al. 2000 Sausages (women 3.5) Meat, meat stews, roasted meat, fish, milk, fat, energy, fiber, Ca Norway Gaard et al. 1996 For distal colon: High intake (≥94 g) of red meat, (women, prospective study: 2.22) Poultry Broiled red meat (1.03), fish. For rectum or proximal colon: red meat Sweden Larsson et al. 2005 For distal colon: Long-term consumption of red meat (1.53), or processed meat (1.50) Poultry, fish USA Chao et al. 2005 Diverse diet with greater proportion of meat, fish, poultry and eggs (men: 1.5); Diverse diet with greater proportion of fruits, vegetables or whole grains Diverse diet USA (California, Minnesota, Utah) F o r P e e r R e v i e w O n l y products (women: 0.40), water (women: 0.55; men: 0.68) Bovine viscera (1.73), cold cuts/sausages (1.47) Lean beef (0.67) Red meat (0.63), total meats (0.70), white meat (0.73), Argentina (Córdoba) Navarro et al. 2003 Red meat; fried or processed meats; fruits, vegetables Finland Pietinen et al. 1999 Processed meat (2.4); offal (2.1); refined cereal products (rice, pasta, pastry), delicatessen, butter, pates (1.7) Fish (1.5); fresh meat (1.2); eggs (1.1); dairy products (1.0) France (Burgundy) Boutron-Ruault et al. 1999 Bread/cereal (1.7); refined sugar (1.4); potatoes (1.2); cakes/desserts (1.1) Vegetables, fruit, fish Meat (white, red or processed), eggs Italy Franceschi et al. 1997 Red meat (1.6) Vitamin C, β-carotene Processed meat Italy (Northern) La Vecchia et al. 1996 (rare/medium done), pan-fried red meat (rare/medium done), fat, alcoholic beverages O n l y White meat (3.29), red meat (1.90), total meat (1.85) Legume (0.53) USA (California) (Seventh Day Adventist) Singh and Fraser 1998 Protein Total calories, total fat, subclasses of fat, carbohydrates, dietary fiber USA (Florida, New York) Kato et al. 1997 Eggs (men: 2.7; women: 2.3), processed meat (men: 2.3), red meat (1.6) Chicken without skin Fat-trimmed red meat, fish; total fat, saturated fat, polyunsaturated fat USA (Hawaii) LeMarchand et al. 1997 grilled/barbecued) microwaved red meat (0.19) meat, grilled/barbecued red meat F o r P e e r R e v i e w Well-/very well-done red meat (fried or Vegetables (0.7), fruit/fruit juice (0.8), Broiled red meat (0.94), total meat, red USA (Maryland) Zheng et al.
		or with greater proportion of refined Red meat (1.03-2.22)	(women: 0.7) Poultry	Fish	Sweden	Larsson et al.
		(1.5)
		grain products (men: 1.7)				2005
		Fresh red meat (women: 2.35), high	Vegetables (0.4)	Fresh red meat (men: 0.89)	Germany	Kampman et al.

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Vegetables, fruit, olive oil, pasta, poultry Italy (Northern) [START_REF] Bosetti | Food groups and risk of squamous cell esophageal cancer in northern Italy[END_REF] Fried