Boeing, H., B. Schlehofer, et al. (1993).
Dietary carcinogens and the risk for glioma and meningioma in Germany.
Int J Cancer 53(4): 561-5.
A population-based case-control study was performed in South-West Germany in 1987/88 with 115 histological confirmed glioma and 81 meningioma cases and 418 randomly selected controls. On the basis of information from a food-frequency questionnaire and questions on food preparation and food supply, the role of dietary carcinogens, in particular N-nitroso compounds or their precursors, on risk for glioma and meningioma were analyzed by multiple logistic regression. Eleven food groups were investigated. The intake of processed meat was significantly associated with an increased risk of glioma. The intake of any food group was not significantly related to meningioma risk. Among single meat products, a significantly higher risk of glioma was found for cooked ham, processed pork meat and fried bacon. For the consumption of 3 N-nitrosamines, assessed from the intake of processed meat and cheese, significant positive relations to glioma risk were found. These N-nitrosamines were also related to meningioma risk, although to a less pronounced extent. The risk for occurrence of glioma was significantly increased for those using vegetable fat frequently for deep frying, as compared with non-users. For the dietary intake of nitrate, nitrite, vitamin C, specific alcoholic beverages, total alcohol, and water from a non-central supply, no elevated risk was found in this study.

DeMarini, D. M., S. B. Hastings, et al. (1997).
Pilot study of free and conjugated urinary mutagenicity during consumption of pan-fried meats: possible modulation by cruciferous vegetables, glutathione S-transferase-M1, and N-acetyltransferase-2.
Mutat Res 381(1): 83-96.

Epidemiological and experimental evidence indicates that consumption of fried meats in conjunction with certain genotypes of phase I and II metabolism genes poses an elevated risk for colorectal cancer. Parallel to this, the consumption of cruciferous vegetables is associated with a reduced risk of colon cancer.
 

Dingley, K. H., K. D. Curtis, et al. (1999).
DNA and protein adduct formation in the colon and blood of humans after exposure to a dietary-relevant dose of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine.
Cancer Epidemiol Biomarkers Prev 8(6): 507-12.

Epidemiology studies have indicated that certain dietary components, including well-cooked meat, are risk determinants for colon cancer. Cooked meat can contain significant quantities of heterocyclic aromatic amines (HCAs), which have been established as carcinogens in laboratory animals. 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is usually the most mass-abundant HCA, with concentrations up to 480 ppb.
The results of this study demonstrate that PhIP is bioavailable to the human colon following defined dietary-relevant doses and forms DNA and protein adducts.

Felton, J. S., M. A. Malfatti, et al. (1997).
Health risks of heterocyclic amines."
 Mutat Res 376(1-2): 37-41.

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 heterocyclic amines. These same compounds produce tumors at multiple organ sites in both mice and rats. One example of these induced tumors has also been seen in nonhuman primates. 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 as is done here, 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 heterocyclic amines could range more than a thousandfold between individuals based on exposure and genetic susceptibility.

Hatch, F. T., M. G. Knize, et al. (1991).
Quantitative structure-activity relationships of heterocyclic amine mutagens formed during the cooking of food.
Environ Mol Mutagen 17(1): 4-19

The major protein-rich foods, particularly muscle meats, contain part-per-billion quantities of potent mutagens formed by frying or broiling to a well-done state. Related mutagens are formed by pyrolysis of amino acids or proteins and in heated model systems. The thermic mutagens so far identified are heterocyclic aromatic amines of aminoimidazo-azaarene (AIA) and aminocarboline classes. The chemicals require activation by enzymes to form metabolites reactive with nucleic acids.

Hatch, F. T., M. G. Knize, et al. (1988).
Cooked-Food Mutagen Reference List and Index.
Environ Mol Mutagen 12 Suppl 14: 1-85.
 

Hatch, F. T., M. G. Knize, et al. (1992).
Quantitative correlation of mutagenic and carcinogenic potencies for heterocyclic amines from cooked foods and additional aromatic amines.
Mutat Res 271(3): 269-87.

Aromatic amines have long been recognized as animal and human carcinogens. Recently heterocyclic aromatic amines (thermic amines) have been found in small amounts in cooked foods, primarily meats, and have proven to be potent mutagens and rodent carcinogens.

Keating, G. A., D. W. Layton, et al. (1999)
Factors determining dietary intakes of heterocyclic amines in cooked foods.
Mutat Res 443(1-2): 149-56.

The identification of heterocyclic amines (HCAs) in cooked foods has focused attention on the potential health effects from their consumption in the diet. Recent studies have estimated daily dietary intakes of HCAs that vary 10-fold and implicated different cooked meats as the prime source of HCAs in the diet. These varied estimates can be attributed to the different dietary assessment methods used in these studies, as well as the different levels of HCAs ascribed to the most commonly consumed cooked meats. Epidemiological studies utilizing information on dietary practice and food intake have found higher risks for several cancers among individuals consuming the highest levels of HCAs. These studies have highlighted the importance of using information on cooking methods in addition to food intake to accurately estimate dietary exposure to HCAs.

Knize, M. G., F. A. Dolbeare, et al. (1994).
Effect of cooking time and temperature on the heterocyclic amine content of fried beef patties.
Food Chem Toxicol 32(7): 595-603.

The mutagenic heterocyclic amines 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (DiMeIQx) were measured in ground-beef patties fried at 150, 190 and 230 degrees C for 2-10 min on each side The amounts of MeIQx, PhIP, DiMeIQx and IQ increased with time and temperature of cooking.
This report shows clearly that increases in cooking temperature and time can have a profound effect on the amounts of heterocyclic amines generated and subsequently consumed in the diet.

Knize, M. G., K. S. Kulp, et al. (2002).
Factors affecting human heterocyclic amine intake and the metabolism of PhIP.
 Mutat Res 506-507: 153-62.

We are working to understand possible human health effects from exposure to heterocyclic amines that are formed in meat during cooking. Laboratory-cooked beef, pork, and chicken are capable of producing tens of nanograms of MeIQx, IFP, and PhIP per gram of meat and smaller amounts of other heteroyclic amines. Well-done restaurant-cooked beef, pork, and chicken may contain PhIP and IFP at concentrations as high as tens of nanograms per gram and MeIQx at levels up to 3 ng/g. 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 beef steak and chicken breast are not significantly different (P=0.36).  Using solid-phase extraction and LC/MS/MS, we quantified four major PhIP metabolites in human urine. In addition to investigating individual variation, we examined the interaction of PhIP with a potentially chemopreventive food. In a preliminary study of the effect of broccoli on PhIP metabolism, we fed chicken to six volunteers before and after eating steamed broccoli daily for 3 days. Preliminary results suggest that broccoli, which contains isothiocyanates shown to induce Phases I and II metabolism in vitro, may affect both the rate of metabolite excretion and the metabolic products of a dietary carcinogen. This newly developed methodology will allow us to assess prevention strategies that reduce the possible risks associated with PhIP exposure.

Knize, M. G., C. P. Salmon, et al. (1999).
Food heating and the formation of heterocyclic aromatic amine and polycyclic aromatic hydrocarbon mutagens/carcinogens.
Adv Exp Med Biol 459: 179-93.

Heterocyclic aromatic amines (HAA) and polycyclic aromatic hydrocarbons (PAH) are mutagens and animal carcinogens sometimes formed when foods are heated or processed. Determining their role in cancer etiology depends on comparing human exposures and determining any significant dose-related effects. Chemical analysis of foods shows that flame-grilling can form both PAH and HAA, and that frying forms predominantly HAA. With detection limits of about 0.1 ng/g, amounts found in commercially processed or restaurant foods range from 0.1 to 14 ng/g for HAA, and levels of PAH up to 1 ng/g in a liquid smoke flavoring. Laboratory fried samples have greater amounts of PAH, up to 38 ng/g in hamburgers, and high levels of HAA, over 300 ng/g, are measured in grilled chicken breast. Understanding the processing conditions that form PAH and HAA can lead to methods to greatly reduce their occurrence in processed foods.

Knize, M. G., R. Sinha, et al. (1995).
Heterocyclic amine content in fast-food meat products.
Food Chem Toxicol 33(7): 545-51.

Heterocyclic aromatic amines are sometimes formed during the cooking of muscle meats, and their mutagenic and carcinogenic effects are of potential concern in the aetiology of human cancer. In a large survey of the heterocyclic amine content of foods, fried or charbroiled hamburgers, fried chicken, chicken breast sandwiches, fish sandwiches and breakfast sausages were purchased from fast-food restaurants. At least three different chains were visited per product and samples from five stores from each chain were pooled. The solid-phase extraction and HPLC method was used to analyse pooled samples for heterocyclic amine content and mutagenic activity with the Ames/Salmonella assay.

Kulp, K. S., M. G. Knize, et al. (2000).
Identification of urine metabolites of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine following consumption of a single cooked chicken meal in humans.
Carcinogenesis 21(11): 2065-72.

 Many studies suggest that mutagenic/carcinogenic chemicals in the diet, like 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), may play a role in human cancer initiation. We have developed a method to quantify PhIP metabolites in human urine and have applied it to samples from female volunteers who had eaten a meal of cooked chicken. For this analysis, urine samples (5 ml) were spiked with a deuterium-labeled internal standard, adsorbed to a macroporous polymeric column and then eluted with methanol.  The variation seen in the total amount, excretion time and metabolite ratios with our method suggests that individual digestion, metabolism and/or other components of the diet may influence the absorption and amounts of metabolic products produced from PhIP.

Layton, D. W., K. T. Bogen, et al. (1995).
Cancer risk of heterocyclic amines in cooked foods: an analysis and implications for research.
 Carcinogenesis 16(1): 39-52.

 Heterocyclic amines (HAs) are formed as pyrolysis products during the cooking of meats/fish. These substances are potent mutagens in the Ames/Salmonella assay and are also carcinogens in laboratory animals. In order to assess the magnitude of the cancer risk posed by their presence in the US diet, we estimated the average intakes of HAs, based on analyses of the concentrations of HAs in cooked foods and data from a dietary survey of the US population and quantified the cancer potencies of the individual compounds using dose-response data from animal bioassays. Measured concentrations of HAs in cooked foods were taken from a major review of the open literature. Only those concentrations that were associated with normal cooking conditions were chosen for use in estimating dietary intakes. The average consumption of HA-bearing foods was determined by analyzing statistically the intakes of 3563 individuals who provided 3 day dietary records in a USDA sponsored random survey of the US population during 1989. Dietary intakes of the five principal HAs in descending order were 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP) > 2-amino-9H-pyrido[2,3-b]indole (A alpha C) > 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) > 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (DiMeIQx) > 2-amino-3-methylimidazo[4,5-f]quinoline (IQ). The carcinogenic potencies, in contrast, were almost the reverse order: IQ > DiMeIQx > MeIQx > PhIP > A alpha C. An upper-bound estimate of the incremental cancer risk is 1.1 x 10(-4), using cancer potencies based on a body surface area basis. Nearly half (46%) of the incremental risk was due to ingestion of PhIP. Consumption of meat and fish products contributed the most (approximately 80%) to total risk.

Nagao, M. and T. Sugimura (1993).
Carcinogenic factors in food with relevance to colon cancer development.
 Mutat Res 290(1): 43-51.

The diet contains various mutagens and carcinogens that can be classified into three groups: naturally occurring chemicals, synthetic compounds and compounds produced by cooking. The first group includes mycotoxins and plant alkaloids while the second is exemplified by food additives and pesticides. The third includes polycyclic aromatic hydrocarbons and heterocyclic amines (HCAs). HCAs are mutagenic to microbes and eukaryotes and their precursors are creatine or creatinine, sugars, and amino acids in meat and fish. Among 10 HCAs so far examined for carcinogenicity in rodents, 2-amino-6-methyldipyrido[1,2-a:3',2'-d]imidazole (Glu-P-1), 2-aminodipyrido[1,2-a:3',2'-d]imidazole (Glu-P-2), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) induced colon cancer in rats. PhIP is an especially interesting compound inducing colon tumors specifically in male F344 rats and only very rarely in females, which develop mammary carcinomas at high frequency instead. Since induced DNA adduct levels, determined by the 32P-postlabeling method, were found to be almost the same in male and female F344 rats adduct formation in itself is not directly responsible for carcinogenesis. We established, however, that PhIP causes increased cell proliferation in colon mucosa but not in the non-target liver or kidney of male rats. Induction of cell proliferation is therefore possibly an additional important factor determining carcinogenic organ specificity. In terms of molecular alteration ras family gene mutations are very rare and no mutations are evident in the p53 gene in colon tumors induced by HCAs. Their development due to HCAs can thus be considered an appropriate experimental model for human colon tumors in which ras or p53 gene activation is not involved. Since HCAs are genotoxic compounds, a causal role in some stage of human colon carcinogenesis is plausible. Exposure to HCAs should accordingly be avoided as far as possible.

Nakagama, H., M. Ochiai, et al. (2002).
A rat colon cancer model induced by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, PhIP." Mutat Res 506-507: 137-44.

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is one of the most abundant heterocyclic amines contained in cooked meat and fish, and induces aberrant crypt foci (ACF), putative preneoplastic lesions of the colon, and colon cancers in male rats when administered orally. As has been reported previously, F344 rats are susceptible to induction of ACF by PhIP, while ACI rats being relatively resistant. Approximately one-fourth of ACF induced by PhIP in F344 rats are dysplastic; exhibiting lesions with structural distortion of the crypt, decrease of goblet cells, nuclear stratification and enlargement of nuclei. Dysplastic ACF demonstrate beta-catenin accumulation, mainly in the cytoplasm, and increased cell proliferation in crypts. These dysplastic ACF are, therefore, strongly considered to be putative preneoplastic lesions of the colon.A genetic trait affecting the susceptibility to colon carcinogenesis in F344 rats was mapped to chromosome 16, between D16Rat17 and D16Wox3, using the number of ACF as a surrogate biomarker for colon carcinogenesis. Since the number of dysplastic lesions is well correlated with the total number of ACF, being approximately one-fourth of the total ACF as described above in F344 rats and will be described elsewhere in ACI rats, the gene involved in the susceptibility to ACF induction may possibly be partly responsible for the susceptibility to colon carcinogenesis by PhIP. We, thus, tentatively referred the name of the candidate susceptibility gene on rat chromosome 16 as susceptibility to colon tumor (Sct).In the present study, the colonic lesions induced by PhIP were well refined histologically and genetically, and the multi-step profiles of colon cancer development by PhIP were well characterized and revealed to be similar to the multi-step model of colon carcinogenesis in humans. The PhIP-induced colon cancer model in rats, thus contributes as a relevant tool to elucidate genetic factors responsible for susceptibility to colon carcinogenesis in human. Other unknown genetic or epigenetic alterations, which are essential for the development of early lesions of colon carcinogenesis, could also be clarified using this system.

Ohgaki, H., S. Takayama, et al. (1991).
Carcinogenicities of heterocyclic amines in cooked food."
Mutat Res 259(3-4): 399-410.

Mutagenic heterocyclic amines in cooked foods were carcinogenic to mice, rats and/or monkeys, when they were given orally continuously. The most common target organ was the liver, but in CDF1 mice lung tumors, forestomach tumors, lymphomas/leukemias, and blood vessel tumors in the brown adipose tissues were also induced. In F344 rats, in addition to liver tumors, tumors in the Zymbal gland, skin, clitoral gland, small and large intestines, oral cavity, and mammary gland were also induced. Monkeys given IQ developed metastatic hepatocellular carcinomas.

Pais, P., C. P. Salmon, et al. (1999).
Formation of mutagenic/carcinogenic heterocyclic amines in dry-heated model systems, meats, and meat drippings.
J Agric Food Chem 47(3): 1098-108.

Pogoda, J. M. and S. Preston-Martin (2001).
Maternal cured meat consumption during pregnancy and risk of paediatric brain tumour in offspring: potentially harmful levels of intake.
 Public Health Nutr 4(2): 183-9.

OBJECTIVE: To describe the relationship between specific levels of nitrite intake from cured meat consumption during pregnancy and the relative risk of paediatric brain tumours in the offspring. DESIGN: Exposure data were previously collected for a population-based case-control study of paediatric brain tumours; data on nitrite content were obtained by a comprehensive literature review of surveys of residual nitrite content in cured meats published in the USA and Canada. The level of nitrite intake for each mother was predicted by year of pregnancy based on survey results. Dose-response was evaluated both categorically and continuously using polynomial and quadratic spline regression. SETTING: The US west coast: Los Angeles County, the San Francisco-Oakland Bay Area and the Seattle-Puget Sound area. SUBJECTS: There were 540 cases diagnosed between 1984 and 1990 at ages varying from 0 to 19 years, and 801 controls frequency-matched by geographic area, age and birth year. RESULTS: In general, survey results suggest a trend of decreasing nitrite levels in cured meats over time. We observed a moderate increase in brain tumour risk in the offspring of mothers with relatively low levels of nitrite consumption from cured meats during pregnancy, and a two- to three-fold risk increase in offspring of mothers who consumed 3 mg day-1 nitrite from cured meats (about 125 g day-1 of cured meat consumption throughout the pregnancy). CONCLUSIONS: A substantial risk of paediatric brain tumour appears to be associated with relatively high levels of maternal cured meat consumption during pregnancy. A more scientifically valid approach than a literature review to estimate nitrite intake from cured meats and data from a large group of highly exposed subjects would be useful in determining potentially harmful levels.

Preston-Martin, S. and B. E. Henderson (1984).
N-nitroso compounds and human intracranial tumours." IARC Sci Publ(57): 887-94.

Experimentalists have shown that various N-nitroso compounds are potent nervous system carcinogens, particularly when animals are exposed transplacentally. Information has been obtained concerning exposure to N-nitroso compounds and their precursors in three case-control studies of intracranial tumour patients in Los Angeles County, California. A study of women (185 pairs) found that level of consumption of nitrite-cured meats was related to meningioma development (p = 0.01). In a similar study of meningiomas in men (105 pairs), the association with cured meats was not clear. The most striking results were obtained in a study of young brain tumour patients (209 matched pairs). Increased risk was associated with maternal contact, during pregnancy, with N-nitrosamine-containing substances, such as burning incense (odds ratio, 3.3; p less than 0.01), sidestream cigarette smoke (odds ratio, 1.5; p = 0.03) and face make-up (odds ratio, 1.6; p = 0.02). Increased risk was also associated with maternal use of diuretics (odds ratio, 2.0; p = 0.03) and antihistamines (odds ratio, 3.4; p less than 0.01) and with the level of maternal consumption of cured meats (p less than 0.01). Diuretics and antihistamines contain nitrosatable amines and amides, and cured meats contain nitrites - chemicals which are precursors of N-nitroso compounds. Additional epidemiological studies of nervous system tumours in young people would appear to offer considerable promise for testing the hypothesis that N-nitroso compounds are etiologically related to human neurogenic neoplasms.

Preston-Martin, S., J. M. Pogoda, et al. (1996).
Maternal consumption of cured meats and vitamins in relation to pediatric brain tumors.
Cancer Epidemiol Biomarkers Prev 5(8): 599-605.

Brain tumors are the leading cause of death from childhood cancer, yet the causes of most of these tumors remain obscure. Few chemicals are effective in causing brain tumors experimentally after systemic administration of low doses; a notable exception is one group of N-nitroso compounds, the nitrosamides (in particular the nitrosoureas). Feeding pregnant animals nitrosamide precursors (e.g., sodium nitrite and an alkylamide such as ethylurea) causes a high incidence of nervous system tumors in offspring. This population-based epidemiological study was designed to test the hypothesis that maternal consumption during pregnancy of meats cured with sodium nitrite increases the risk of brain tumors among offspring. The intake of vitamins C and E blocks endogenous formation of nitroso compounds and was expected to be protective. Mothers of 540 children under age 20 with a primary brain tumor diagnosed during 1984-1991 and 801 control children in the same 19 counties on the U.S. West Coast were interviewed. Risk increased with increasing frequency of eating processed meats [odds ratio (OR) = 2.1 for eating at least twice a day compared to not eating; 95% confidence interval (CI) = 1.3-3.2; P = 0.003). Risk also increased with increasing average daily grams of cured meats or mg of nitrite from cured meats (P for each <0.005) but not with nitrate from vegetables. Daily use of prenatal vitamins throughout the pregnancy decreased risk (OR = 0.54; CI = 0.39-0.75). Risk among mothers who consumed above the median level of nitrite from cured meat was greater if vitamins were not taken (OR = 2.4; CI = 1.4-3.6) than if they were (OR = 1.3). These effects were evident for each of three major histological types and across social classes, age groups, and geographic areas. This largest study to date of maternal diet and childhood brain tumors suggests that exposure during gestation to endogenously formed nitroso compounds may be associated with tumor occurrence. Laboratory exploration is needed to: (a) define dietary sources of exposure to alkylamides; (b) investigate the reactivity of nitrite in high concentration such as around bits of cured meats in the stomach after ingestion compared to nitrite in dilute solution; and (c) confirm that simultaneous ingestion of alkylamides and cured meats leads to the endogenous formation of nitrosamides.

Preston-Martin, S., M. C. Yu, et al. (1982).
N-Nitroso compounds and childhood brain tumors: a case-control study.
 Cancer Res 42(12): 5240-5

We questioned mothers of 209 young brain tumor patients and mothers of 209 controls about experiences of possible etiological relevance which they had during pregnancy or which their children had while growing up. Long-suspected brain tumor risk factors such as head trauma and X-rays appeared to be factors for relatively few cases. Increased risk was associated with maternal contact with nitrosamine-containing substances such as burning incense (odds ratio, 3.3; p = 0.005), sidestream cigarette smoke (odds ratio, 1.5; p = 0.03), and face makeup (odds ratio, 1.6; p = 0.02); with maternal use of diuretics (odds ratio, 2.0; p = 0.03) and antihistamines (odds ratio, 3.4; p = 0.002); and with the level of maternal consumption of cured meats (p = 0.008). These drugs contain nitrosatable amines and amides, and the cured meats contain nitrites, chemicals which are precursors of N-nitroso compounds. We propose a hypothesis that brain tumors in these young people are related to in utero exposure to N-nitroso compounds and their precursors, the most potent nervous system carcinogens known in experimental animals.

Salmon, C. P., M. G. Knize, et al. (2000).
Minimization of heterocyclic amines and thermal inactivation of Escherichia coli in fried ground beef.
 J Natl Cancer Inst 92(21): 1773-8.

BACKGROUND: Heterocyclic amine carcinogens are formed during the cooking of a number of foods, especially well-done meats. Lower temperatures and shorter cooking times can minimize the formation of these carcinogens, yet a major food safety concern is that pathogens in the meat must be thermally inactivated. This study investigated cooking techniques that minimize heterocyclic amine formation while simultaneously destroying contaminating bacteria. METHODS: Ground beef patties were inoculated with Escherichia coli K12 bacteria and fried to internal temperatures ranging from 35 degrees C to 70 degrees C in a skillet preheated to 160 degrees C, 180 degrees C, or 200 degrees C. Each patty was then analyzed for four common heterocyclic amines and for surviving bacteria. Additionally, the frequency of turning of the beef patty during cooking was varied (a single turn or multiple turns), length of time required for each patty to reach 70 degrees C was recorded, and heterocyclic amine levels were determined. An additional pan temperature of 250 degrees C was tested for its effect on heterocyclic amine formation but not on bacterial killing. Statistical tests were two-sided. RESULTS: Colony-forming bacteria were reduced by five orders of magnitude at internal temperatures greater than 60 degrees C, regardless of cooking method, and were completely inactivated at 70 degrees C. For patties turned just once, heterocyclic amine levels increased as the cooking temperatures increased. However, levels of heterocyclic amines were statistically significantly lower with turning every minute. For each pan temperature, patties reached 70 degrees C internal temperature sooner when they were turned every minute than when they were turned just once during cooking. CONCLUSION: Lowering the pan temperature and turning the patties frequently can greatly reduce the formation of heterocyclic amines and can simultaneously achieve bacterial inactivation with little or no increase in cooking time, ensuring a product that is safe for human consumption.

Sinha, R., M. G. Knize, et al. (1998).
Heterocyclic amine content of pork products cooked by different methods and to varying degrees of doneness." Food Chem Toxicol 36(4): 289-97.

Heterocyclic amines (HCAs) are known mutagens and animal carcinogens produced in meats cooked at high temperature. As pork is the second most frequently consumed meat in the United States, five predominant HCAs [2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-3,4,8-trimethylimidazo[4.5-f]quinoxaline (DiMeIQx) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)] were measured in various pork products, cooked by different techniques and to varying doneness levels. Pork chops and ham slices were pan-fried and oven-broiled; bacon was pan-fried, oven-broiled or microwaved; hot dogs were pan-fried, oven-broiled, grilled/barbecued or boiled; sausage links and patties were pan-fried. All the products were cooked to three levels of doneness: just until done, well done or very well done. HCA type and level varied substantially by pork product, cooking method and doneness level. The highest PhIP levels were found in well done and very well done oven-broiled bacon; for very well done 30.3 and 4.0 ng per gram of meat of PhIP and MeIQx, respectively. Pan-fried very well done sausage patties contained 5.4 ng of MeIQx per gram of meat, while sausage links contained 1.3 ng per gram of meat. MeIQx was formed in well done and very well done pan-fried but not broiled pork chops. Hot dogs or ham slices had low or undetectable levels of HCAs. These results demonstrate that epidemiological studies investigating the relationship between HCA intake and cancer risk need to incorporate type of meat, cooking method and degree of doneness/surface browning into questions to assess adequately an individual's HCA exposure.

Sinha, R., N. Rothman, et al. (1995).
High concentrations of the carcinogen 2-amino-1-methyl-6-phenylimidazo- [4,5-b]pyridine (PhIP) occur in chicken but are dependent on the cooking method.
 Cancer Res 55(20): 4516-9.

Heterocyclic aromatic amines (HAAs) are mutagenic and carcinogenic compounds found in meats cooked at high temperatures. Although chicken is consumed in large quantities in the United States, there is little information on its HAA content. The objective of this study was to measure the five predominant HAAs (IQ, MeIQ, MeIQx, DiMeIQx, and PhIP) in chicken cooked by various methods to different degrees of doneness. Chicken breasts were panfried, oven-broiled, or grilled/barbecued. Whole chickens were roasted or stewed. Skinless, boneless chicken breasts were cooked to three degrees of doneness: just until done, well done, or very well done. High levels of PhIP (ranging from 12 to 480 ng/g cooked meat) were found in chicken breasts when panfried, oven-broiled, and grilled/barbecued but not in while roasted or stewed chicken. PhIP concentration increased in skinless, boneless chicken breast with longer cooking time, higher internal temperature, and greater degree of surface browning. PhIP concentration was also high in chicken breasts cooked with skin and bones. MeIQx and DiMeIQx levels increased with the degree of doneness, whereas IQ and MeIQ were not detectable in any of these chicken samples. Certain cooking methods produce PhIP, a known colon and breast carcinogen in rodents and possibly a human carcinogen, at substantially higher levels in chicken than has been reported previously in red meat.

Sinha, R., N. Rothman, et al. (1998).
Heterocyclic amine content in beef cooked by different methods to varying degrees of doneness and gravy made from meat drippings.
Food Chem Toxicol 36(4): 279-87.

Meats cooked at high temperatures sometimes contain heterocyclic amines (HCAs) that are known mutagens and animal carcinogens, but their carcinogenic potential in humans has not been established. To investigate the association between HCAs and cancer, sources of exposure to these compounds need to be determined. Beef is the most frequently consumed meat in the United States and for this study we determined HCA values in beef samples cooked in ways to represent US cooking practices, the results of which can be used in epidemiological studies to estimate HCA exposure from dietary questionnaires. We measured five HCAs [2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (DiMeIQx) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)] in different types of cooked beef using solid-phase extraction and HPLC. Steak and hamburger patties were pan-fried, oven-broiled, and grilled/barbecued to four levels of doneness (rare, medium, well done or very well done), while beef roasts were oven cooked to three levels of doneness (rare, medium or well done). The measured values of the specific HCAs varied with the cut of beef, cooking method, and doneness level. In general, MeIQx content increased with doneness under each cooking condition for steak and hamburger patties, up to 8.2 ng/g. PhIP was the predominant HCA produced in steak (1.9 to 30 ng/g), but was formed only in very well done fried or grilled hamburger. DiMeIQx was found in trace levels in pan-fried steaks only, while IQ and MeIQ were not detectable in any of the samples. Roast beef did not contain any of the HCAs, but the gravy made from the drippings from well done roasts had 2 ng/g of PhIP and 7 ng/g of MeIQx. Epidemiological studies need to consider the type of meat, cooking method and degree of doneness/surface browning in survey questions to adequately assess an individual's exposure to HCAs.

Snyderwine, E. G., R. Sinha, et al. (2002).
Highlights of the eighth international conference on carcinogenic/mutagenic N-substituted aryl compounds.
Mutat Res 506-507: 1-8.

Research in the 20th century initially identified arylamines as causative factors in occupational carcinogenesis, especially bladder cancer, and subsequently identified arylamines as a major class of mutagens/carcinogens in the environment and diet that are potential risk factors in a variety of human cancers. Current research focuses on understanding of mechanisms of arylamine carcinogenesis, such as the role of metabolic processing, DNA adduct formation, and mutagenesis, and learning more about the molecular alterations in carcinomas induced by these compounds. Furthermore, research to identify human exposures, including developing more sensitive methods for analyzing environmental samples and identifying suitable biomarkers are important aspects of contemporary investigations. In addition, better evaluation of the risk of these compounds in human cancer especially with regard to the impact of genetic polymorphisms is a major focus of research in this field. Although current population studies have sometimes been described as equivocal, improved tools for epidemiology, refined human biomonitoring methods and collaborative endeavors to study multiple population groups now provide a better means to ultimately define the role of arylamines in human carcinogenesis. The purpose of the Eighth International Conference on Carcinogenic/Mutagenic N-Substituted Aryl Compounds, held in Washington, DC, 12-14 November 2001, was to explore the current scope of studies on arylamine carcinogenesis among scientists in basic research and epidemiology and to discuss future research priorities. With the intent of providing a view to the current field of research on aromatic amines, this review presents a synopsis of the Proceedings of the Eighth International Conference and highlights the manuscripts contained in this special issue of Mutation Research.

Snyderwine, E. G., R. J. Turesky, et al. (1997).
Metabolism of food-derived heterocyclic amines in nonhuman primates.
 Mutat Res 376(1-2): 203-10.

During the cooking of meats, several highly mutagenic heterocyclic amines (HCAs) are produced. Three HCAs, IQ, MeIQx, and PhIP have been under study for carcinogenicity in cynomolgus monkeys, and to date, IQ 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 results from these studies support the importance of N-hydroxylation in the carcinogenicity of HCAs in nonhuman primates and by analogy, the importance of this metabolic activation step in the possible carcinogenicity of dietary HCAs in humans.

Vanderlaan, M., B. E. Watkins, et al. (1988).
Monoclonal antibodies for the immunoassay of mutagenic compounds produced by cooking beef.
 Carcinogenesis 9(1): 153-60.

A family of 2-amino-N-methylimidazoazaarene (AIA) mutagens are produced in meats by cooking, and are of concern as potential carcinogens in the human diet. These are potent genotoxins in bacterial mutation assays, but are present only in trace quantities in cooked foods. Conventional analytical chemistry methods have allowed us to identify six members of the AIA class, but these methods are much too labor-intensive for us to quantify mutagens in large numbers of meat samples. To improve the assay of these mutagens we have developed six monoclonal antibodies (named IQ-1, IQ-2, AIA-1, AIA-2, AIA-4, and AIA-7) and demonstrated their utility in an immunoassay for cooked food mutagens. Each antibody has its own unique binding selectivity pattern; some are compound-specific, and some class-specific. A comparison was made between cooked beef extracts that differed 200-fold in mutagenic activity. The high-mutagen extract had significantly more material that was immunologically cross-reactive with the anti-AIA antibodies than did the low-mutagen extract. Taken as a set, these antibodies will allow rapid quantitation of foods for several AIA mutagens, and will aid in the isolation and characterization of other AIAs and AIA-metabolites.

Wakabayashi, K., Y. Totsuka, et al. (1997).
Human exposure to mutagenic/carcinogenic heterocyclic amines and comutagenic beta-carbolines.
 Mutat Res 376(1-2): 253-9.

Various kinds of mutagenic and carcinogenic heterocyclic amines (HCAs) are produced by heating protein-rich foods, such as meat and fish. To evaluate the risk of these HCAs in terms of human cancer development, exposure levels must be measured. We therefore analyzed their amounts in various kinds of cooked foods and in urine samples of healthy volunteers living in Tokyo.

Yano, M., K. Wakabayashi, et al. (1988).
Presence of nitrosable mutagen precursors in cooked meat and fish."
 Mutat Res 202(1): 119-23.

 Broiled chicken, pork, mutton, beef and sun-dried sardine were found to yield direct-acting mutagenicity after nitrite treatment. When 50% methanol extracts of cooked foods were treated with 50 mM nitrite at pH 3 for 1 h at 37 degrees C, they induced 3800-17,900 revertants of Salmonella typhimurium TA100 and 15,000-43,600 revertants of TA98 per g. In contrast, raw meat and uncooked sun-dried sardine showed little or no mutagenicity after nitrite treatment. Treatment of broiled chicken with 0.5-3 mM nitrite, which is a physiologically feasible concentration in the human stomach under some conditions, induced direct-acting mutagenicity. When broiled chicken was treated with 1 mM nitrite at pH 3 for 1 h at 37 degrees C, its mutagenicities on TA100 and TA98 without S9 mix were 7100 and 5400 revertants/g,