Potential Sources of Carcinogenic Heterocyclic Amines in the Chinese Diet: Results from a 24-H Dietary Recall Study in Singapore

ORIGINAL COMMUNICATION Potential sources of carcinogenic heterocyclic amines in the Chinese diet: results from a 24-h dietary recall study in Singapore

W-P Koh1, HN Yang1, HQ Yang1, S-H Low1 and A Seow1*

1Department of Community, Occupational and Family Medicine, National University of Singapore, Singapore

Objective: Heterocyclic amines (HCAs) from high-temperature cooking of meat have been linked to increased cancer incidence in Western populations, but data on the sources of HCAs in Asian diets are scarce. Our aim was to identify potential sources of HCAs in the Chinese diet, and to provide the basis for efforts to quantify dietary exposure to these compounds. Design and setting: We conducted 24-h dietary recall interviews among 986 Chinese men and women in Singapore, who were a randomly selected subpopulation of participants from the Singapore Chinese Health Study, a population-based cohort. Details of all foods and beverages consumed by each subject in the past 24 h were recorded, and information on meat type, cooking method and portion size were abstracted from all meat-containing dishes, and gram weight equivalents computed. Results: The mean meat intake per person was 103.0 g/day (standard deviation 74.2), of which 97.2% was fresh meat. Fish (38.0%), pork (30.6%), and poultry (21.0%) accounted for 89.6% of meat consumed. Patterns of meat consumption and cooking methods differed markedly from Western populations. Documented high-temperature cooking methods, combined with stir-frying, accounted for 44.3% of fish, 35.1% of pork and 25.6% of poultry consumed. Specifically, potentially significant sources of HCAs were pan-fried fish and barbecued pork. Conclusions: Our results identify the potential sources of HCA in the Chinese diet, highlight aspects which are relevant to HCA formation and intake, and call for novel approaches to estimating individual exposure to dietary HCAs in this and similar populations. European Journal of Clinical Nutrition (2005) 59, 16–23. doi:10.1038/sj.ejcn.1602027 Published online 25 August 2004

Keywords: heterocyclic amines; meat; cancer risk; 24-h recall; dietary intake

Introduction 6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-amino-3,8-di- The food-derived heterocyclic amines (HCAs) comprise a methylimidazo[4,5-f]quinoxaline (MeIQx), are recognized as family of mutagenic/carcinogenic compounds formed from potent experimental mutagens and carcinogens capable of creatin(in)e, amino acids and sugars in muscle meats by inducing tumors in multiple tissue sites in several animal cooking meats at high temperatures using ordinary household species (Hasegawa et al, 1994; Yoshimoto et al, 1999; Archer et al, methods (Jagerstad et al, 1984). Today approximately 20 HCAs 2000; Nagao et al, 2002). Recently, there has been a growing have been identified and several, including 2-amino-1-methyl- body of epidemiological evidence supporting the relationship between intake of dietary HCAs and increased risk of certain cancers of major importance in many countries, such as the *Correspondence: A Seow, Department of Community, Occupational and colo-rectum (Gerhardsson de Verdier et al, 1991; Lang et al, Family Medicine, National University of Singapore, MD3, 16 Medical 1994; Kampman et al, 1999; Wu et al, 2001; Murtaugh et al, Drive, Singapore 117597, Singapore. 2004), lung (Deneo-Pellegrini et al, 1996; Sinha et al, 1998a), E-mail: [email protected] Guarantors: W-P Koh and A Seow. pancreas (Anderson et al, 2002) and breast (Knekt et al,1990;De Contributors: W-PK and AS are co-principal investigators, and were Stefani et al, 1997; Zheng et al, 1998; Sinha et al, 2000). responsible for writing the manuscript; S-HL trained the interviewers HCAs have been detected at parts per billion (ng/g) and supervised the fieldwork for the Singapore Chinese Health Study; concentrations in cooked meat and the most important HNY and HQY abstracted and analyzed the data. Received 30 March 2004; revised 28 May 2004; accepted 7 June 2004; factors influencing their formation in cooking are meat published online 25 August 2004 type, cooking method, temperature and time (Sinha et al, Heterocyclic amines in the Chinese diet W-P Koh et al 17 1995, 1998b; Skog et al, 1998; Keating et al, 1999). Differ- followed a written protocol to ensure uniformity in eliciting ences in methods of preparing meat before cooking, the use and recording all food and beverage items consumed within of marinade or type of cooking oil may also affect the and outside the home over the last 24 h. A total of 986- formation of HCAs in cooked meat (Knize et al, 1995; Layton person days were recorded with 293 (29.7%) being weekends et al, 1995; Monti et al, 2001). Exposure to HCAs therefore (Saturday and Sunday). varies greatly between populations, depending on dietary For each food item consumed, the name of the dish, type preferences and cooking methods. An understanding of the of cooking method used (stir-fried, boiled/steamed, deep- potential effect of HCAs on cancer risk in diverse populations fried, pan-fried, roasted/grilled, stewed), ingredients and requires an instrument capable of measuring exposure to portion size of each ingredient were elicited. Most Chinese these compounds in diets specific to that population. Such dishes are mixed dishes with reasonably standard recipes. an instrument would comprise a questionnaire which The cooking methods are also easily distinguishable, and addresses frequency of intake, type of meat and cooking participants did not encounter any difficulties in describing methods, and reliable estimates of HCA concentrations in these methods for food consumed either inside or outside meats cooked according to representative cooking practices the home. Portion sizes were recorded as multiples of used in that population (Sinha & Rothman, 1997; Keating common household measurements, and the interviewers et al, 1999). Such questionnaires have been developed and carried a set of standard household utensils to help the applied in various populations in the West (Layton et al, subjects indicate the actual amount eaten (eg half a rice-bowl 1995; Augustsson et al, 1997; Byrne et al, 1998; Norrish et al, of sliced pork, one Chinese spoonful of dried mushrooms). 1999) and in Japan (Kobayashi et al, 2002), as a means of After the interview, all items in the recall were coded and estimating exposure of HCA in the usual diet of these gram weights assigned according to the Singapore Chinese cultures, as well as in epidemiologic studies of the relation- Food Composition Database (Hankin et al, 2001). Based on ship between HCA intake and cancer risk (Sinha et al, 1998a; these data, we selected all meat items (including fresh meat, Zheng et al, 1998; Norrish et al, 1999). To our knowledge, processed meat and meat products, see definitions below) such an instrument has yet to be developed for the Chinese consumed by the respondents and categorized them accord- diet, and analytical data on HCA concentrations in meat, ing to type of meat (pork, chicken, etc) and cooking method in the form commonly consumed by these populations, is (boiling, pan-frying, stir-frying, etc). Where cooking meth- lacking. ods were specific to the form of meat (eg pork ribs and In this study, we describe the pattern of meat consumption minced pork are commonly cooked using different meth- and specific cooking methods among middle-aged and older ods), we distinguished between specific forms of meat for Chinese men and women in Singapore. clarity. In this way, the gram weight equivalents of each type of meat by different cooking methods consumed in the past 24 h were obtained for each subject. Methods Study population Data were obtained from 24-h dietary recall interviews Definition of cooking method and meat type conducted among a random sample of participants of the The definitions of the various cooking methods are listed in Singapore Chinese Health Study. The latter is an ongoing Table 1. In the analysis, we also regrouped these methods prospective cohort of 63 257 healthy Chinese men and according to the potential to generate HCAs based on women (Hokkien and Cantonese in dialectal origin) who cooking temperatures cited in the literature. Hence, steam- are citizens or permanent residents living in public housing ing and boiling were grouped as ‘low’ temperature methods; estates (these provide housing for close to 90% of the stewing and deep-frying as ‘medium’; pan-frying, barbecu- population). The cohort was recruited between 1993 and ing/open roasting and grilling as ‘high’ temperature methods 1998, and all members completed at baseline a food (Skog, 1993; Robbana-Barnat et al, 1996; Knize et al, 1999). frequency questionnaire. As part of the validation study, a Stir-frying was analyzed separately as there is insufficient random sample of participants was selected to complete two data in the literature on the range of temperature achieved 24-h recall interviews covering a weekday and a weekend. during cooking with this method. We also examined fresh The current data is based on the first of these 24-h recall and processed meat intake separately. In summing up intake interviews from the 986 cohort members who represent a of fresh meat, we excluded organ meats, for example, liver randomly selected 3% of the study population recruited and kidneys, since these foods contain little creatine and between February 1993 and August 1996 (Hankin et al, 2001). therefore have relatively low concentrations of HCA when cooked (Robbana-Barnat et al, 1996). We also excluded meat products such as prepackaged meat-balls and fish-balls, which Data collection contain a high proportion, by weight, of flour. Processed All interviews were conducted at participants’ homes meats in this analysis refers to foods that have been between April 1994 and March 1997, by trained interviewers. precooked, preserved or mixed with other foods prior to the Each interview lasted about 25–30 min and the interviewers packaging by the manufacturer. Some examples are Chinese

European Journal of Clinical Nutrition Heterocyclic amines in the Chinese diet W-P Koh et al 18 Table 1 Definition of terms used to describe cooking methods in the Singapore Chinese Health Study. From the list of 1421 study subjects, 986 agreed to participate, representing a response Cooking method Definition rate of 69%. There were a total of 400 men (40.6%) and 586 women (59.4%), with a dialect distribution of 609 (61.8%) Steamed/boiled Cooked over steam or in boiling water Hokkiens and 377 (38.2%) Cantonese. The age range was Stir-fried Tossed quickly in a thin layer of preheated 45–74 y, and the mean age was 58.0 (standard deviation s.d. oil till the food is cooked Open roasted/grilled Cooked by direct exposure to radiant heat 8.2) y. These distributions were very similar to the age and over a grill or through the flames of naked gender distribution in the original cohort (mean age 56.5 y, fire s.d. 8.0 y, 44.2% men and 55.8% women), but there was a Deep-fried Immersed in preheated oil or frying fat higher proportion of Hokkiens (who comprised 53.7% in the Stewed Fried in a small amount of oil till slightly browned, after which liquid is added, parent population). The study population in this paper was brought to the boil and allowed to simmer also very similar to the parent population in the distribution Pan-fried Fried in a thin layer of oil and turned once of educational level (27% in both study populations had no or twice as each side is cooked formal education), marital status (85% in the current study vs 83% in the parent population were married and living with their spouses), birth place (71% in the current study vs sausages, luncheon meat, bacon slices, Western sausages, 73% in parent study were born in Singapore) and length of canned sardines and tuna, salted fish, dried shrimps, dried residence in Singapore (98% in both populations had lived in oysters or mussels, dried scallops and dried cuttlefish. Singapore for 25 y or more). The mean intake of meat among the 986 subjects was 103.0 (s.d. 74.2) g/day. Fresh or nonprocessed meat con- Statistical methods stituted 97.2% by weight of all meats consumed. Table 2 Differences in meat intake between gender and dialect shows the mean daily intake per person (g/day), standard groups were tested using the Mann–Whitney U-test. All error of the mean and the percentage of total meat intake of P-values reported are two-sided, and P-values of o0.05 were each type of meat by gender and dialect group among the considered statistically significant. All analyses were per- 986 subjects. Fish, pork, poultry (chicken and duck) and formed using the SPSS 11.5 statistical package (SPSS Inc., crustaceans (primarily prawns/shrimp and cuttlefish) ac- Chicago, IL, USA). counted for 98.7% of all meats consumed. Among these, fish and pork were the two most common types of meat in both dialect groups and genders, constituting 38.0 and 30.6%, Results respectively, among all meats in this study population. This study population represents a randomly generated Poultry constituted 21.0%, the majority of the poultry being subpopulation of the cohort of men and women in the chicken (78% chicken vs 22% duck). Conversely, other meats

Table 2 Intake of fresh and processed meat by gender and dialect group among 986 Singapore Chinese men and women

Male (n ¼ 400) Female (n ¼ 586)

Hokkien (n ¼ 262) Cantonese (n ¼ 138) Hokkien (n ¼ 347) Cantonese (n ¼ 239)

Meana s.e.m.b (%)c Mean s.e.m. (%) Mean s.e.m. (%) Mean s.e.m. (%)

Fresh meat Fish 42.1 3.0 (36.1) 40.6 4.4 (30.8) 35.4 2.4 (41.5) 35.7 3.5 (36.8) Pork 37.7 2.7 (32.4) 40.1 3.5 (30.4) 24.2 1.9 (28.3) 28.3 2.3 (29.2) Poultry 20.2 2.5 (17.3) 37.5 5.1 (28.4) 15.6 2.0 (18.3) 22.3 2.7 (23.0) Crustaceans 11.0 1.4 (9.4) 8.2 1.5 (6.2) 7.6 1.0 (8.9) 6.0 1.0 (6.2) Beef/lamb/mutton 1.7 0.9 (1.5) 1.7 1.0 (1.3) 0.6 0.4 (0.7) 0.4 0.2 (0.4) Other meat 0.2 0.2 (0.2) 0.0 0.0 0.0 0.1 0.1 (0.1) 1.1 0.8 (1.1)

Processed meat Fish 1.7 0.4 (1.5) 1.5 0.5 (1.1) 0.8 0.2 (0.9) 0.7 0.2 (0.7) Crustaceans 1.1 0.2 (0.9) 1.4 0.5 (1.1) 1.0 0.2 (1.2) 1.3 0.5 (1.3) Pork 0.8 0.4 (0.7) 0.7 0.4 (0.5) 0.1 0.1 (0.1) 1.1 0.4 (1.1) Poultry 0.0 0.0 (0.0) 0.2 0.2 (0.2) 0.0 0.0 (0.0) 0.2 0.2 (0.2)

Total meat 116.5 4.5 (100.0) 131.9 7.4 (100.0) 85.4 3.5 (100.0) 97.1 4.8 (100.0)

aMean intake in g/day. bStandard error of the mean. cPercentage of total meat intake.

European Journal of Clinical Nutrition Heterocyclic amines in the Chinese diet W-P Koh et al 19 like beef, lamb and mutton, which are commonly consumed relative contribution within each meat type. The main in the Western populations, only constituted about 1% of all cooking methods for each type of meat were fairly similar meats among the subjects. among the four gender-dialect groups studied. Boiling or The mean intake of meat for men (121.8 g/day) was steaming was the most common cooking method in this significantly higher than that of women (90.1 g/day, population for fish (45.6%), pork (48.2%) and poultry Po0.001). This difference extended to all meat types, (37.9%) by weight. Stir-frying was the most common particularly pork (Po0.001), fish (P ¼ 0.010) and poultry method of cooking crustaceans, commonly prawns (P ¼ 0.053). In general, meat intake among Cantonese was (45.9%), and was used to cook 18.5% of all pork and 13.5% higher than among Hokkiens in both genders (P ¼ 0.096), of all poultry by weight. Pork and poultry were also cooked the main difference being in the intake of poultry (chicken by open roasting/barbecuing or grilling; these methods and duck), which was markedly higher among Cantonese accounted for a mean intake of 6.6 g of meat per day for (Po0.001). each subject in our study population. Figure 1 shows the Table 3 describes the mean daily intake per person (g/day), distribution by cooking method of the four most common standard error of the mean and percentage of total meat meat types in this population. Among all study subjects intake of the various meats by cooking method, and their combined, intake by weight of fish was highest, and 44.3%

Table 3 Meat intake by type, cooking method, gender and dialect group among 986 Singapore Chinese men and women

Male (n ¼ 400) Female (n ¼ 586)

Hokkien (n ¼ 262) Cantonese (n ¼ 138) Hokkien (n ¼ 347) Cantonese (n ¼ 239)

Meana s.e.m.b (%)c Mean s.e.m. (%) Mean s.e.m. (%) Mean s.e.m. (%)

Fish Boiled/steamed 21.6 2.8 (49.3) 21.3 4.0 (50.6) 13.3 1.6 (36.8) 18.3 2.4 (50.3) Pan-fried 17.8 1.7 (40.6) 15.2 2.6 (36.1) 18.6 1.8 (51.4) 16.0 2.8 (44.0) Stir-fried 1.3 0.5 (3.0) 1.9 0.8 (4.5) 1.5 0.4 (4.1) 0.7 0.3 (1.9) Deep-fried 1.4 0.6 (3.2) 2.2 1.2 (5.2) 2.0 0.7 (5.5) 0.7 0.5 (1.9) Processed 1.7 0.4 (3.9) 1.5 0.5 (3.6) 0.8 0.2 (2.2) 0.7 0.2 (1.9) Total 43.8 3.1 (100.0) 42.1 4.4 (100.0) 36.2 2.4 (100.0) 36.4 3.5 (100.0)

Pork Boiled/steamed 18.5 1.7 (48.1) 20.1 2.4 (49.3) 12.6 1.3 (51.9) 12.5 1.5 (42.5) Stir-fried 6.1 0.9 (15.8) 7.1 1.3 (17.4) 6.0 0.7 (24.7) 4.7 0.9 (16.0) Barbecued/open roasted 5.6 1.1 (14.5) 6.5 1.8 (15.9) 1.9 0.4 (7.8) 4.2 0.9 (14.3) Stewed 3.9 1.0 (10.1) 3.6 1.3 (8.8) 1.9 0.5 (7.8) 3.3 1.1 (11.2) Deep-fried 2.1 0.6 (5.5) 0.3 0.2 (0.7) 0.3 0.1 (1.2) 2.3 0.9 (7.8) Pan-fried 0.9 0.4 (2.3) 2.4 1.0 (5.9) 1.0 0.3 (4.1) 0.9 0.5 (3.1) Processed 0.8 0.4 (2.1) 0.7 0.4 (1.7) 0.1 0.1 (0.4) 1.1 0.4 (3.7) Other 0.6 0.3 (1.6) 0.1 0.1 (0.3) 0.5 0.2 (2.1) 0.4 0.2 (1.4) Total 38.5 2.8 (100.0) 40.8 3.5 (100.0) 24.3 1.9 (100.0) 29.4 2.4 (100.0)

Poultry Boiled/steamed 7.6 1.4 (37.8) 14.9 2.9 (39.5) 4.7 1.3 (30.1) 10.0 2.0 (44.5) Stewed 5.7 1.2 (28.3) 9.5 3.3 (25.2) 2.1 0.6 (13.5) 5.9 1.5 (26.2) Stir-fried 3.2 1.3 (15.9) 1.5 0.7 (4.0) 3.4 0.7 (21.8) 2.8 0.8 (12.4) Deep-fried 1.8 0.8 (9.0) 4.9 1.5 (13.0) 3.2 1.2 (20.5) 1.2 0.4 (5.3) Open roasted/grilled 1.6 0.7 (8.0) 6.0 2.0 (15.9) 1.7 0.6 (10.9) 2.4 1.0 (10.7) Pan-fried 0.2 0.2 (1.0) 0.0 0.0 (0.0) 0.3 0.2 (1.9) 0.0 0.0 (0.0) Other 0.0 0.0 (0.0) 0.9 0.7 (2.4) 0.2 0.2 (1.3) 0.2 0.2 (0.9) Total 20.1 2.5 (100.0) 37.7 5.1 (100.0) 15.6 2.0 (100.0) 22.5 2.7 (100.0)

Seafood Stir-fried 5.7 0.9 (47.1) 2.6 0.8 (27.1) 4.2 0.7 (48.8) 3.8 0.7 (52.1) Boiled/steamed 3.4 0.6 (28.1) 4.7 1.2 (48.9) 2.3 0.7 (26.8) 1.5 0.4 (20.5) Deep-fried 0.8 0.5 (6.6) 0.7 0.4 (7.3) 0.3 0.2 (3.5) 0.3 0.2 (4.1) Pan-fried 0.2 0.2 (1.7) 0.0 0.0 (0.0) 0.2 0.1 (2.3) 0.0 0.0 (0.0) Stewed 0.4 0.2 (3.3) 0.0 0.0 (0.0) 0.2 0.2 (2.3) 0.4 0.2 (5.5) Other 0.5 0.3 (4.1) 0.2 0.2 (2.1) 0.4 0.2 (4.7) 0.0 0.0 (0.0) Processed 1.1 0.2 (9.1) 1.4 0.5 (14.6) 1.0 0.2 (11.6) 1.3 0.5 (17.8) Total 12.1 1.4 (100.0) 9.6 1.8 (100.0) 8.6 1.1 (100.0) 7.3 1.1 (100.0) aMean intake in g/day. bStandard error of the mean. cPercentage of total meat intake.

European Journal of Clinical Nutrition Heterocyclic amines in the Chinese diet W-P Koh et al 20 45 not otherwise specified (includes processed meat) all meats from the neighboring countries, and common 40 stir-fried meats such as chicken, fish, seafood, pork, beef and mutton 35 high: pan-frying/open roasting/grilling are freely available in the markets located in all residential moderate: stewing/deep-frying 30 estates. Most middle- and older-aged Chinese are either first- low: steaming/boiling 25 or second-generation Singaporeans. While many younger 20 Chinese may consume a diet that is more ‘Westernized’, the 15 older Chinese still retain closely the eating habits associated 10 with their Chinese roots, in this case, from Fujian and

GRAMS PER PERSON DAY 5 Guangzhou, both in the southern part of the Chinese

0 mainland. This is also inferred from the present study, in PORK FISH POULTRY CRUSTACEANS which very few of the 986 subjects reported having MEAT TYPE consumed traditionally ‘Western’ foods such as hamburger Figure 1 Intake of meat by cooking method (weight in grams per (one person), bacon (one person), Western sausages (three person per day) among 986 Singapore Chinese men and women. persons) and beef steak (zero persons) in their 24-h dietary recalls. Boiling and steaming, and stir-frying are character- istic of the regional cuisine in this area, and may explain the of fish by weight was also most likely to be cooked using propensity towards these cooking methods. In addition, a high-temperature methods (specifically, pan-frying) large proportion of Chinese in Singapore are Buddhists or (Figure 1). Documented high-temperature methods (open Taoists, and strict adherents may choose to abstain from beef roasting/barbecuing, grilling, pan-frying), combined with as part of their religious practice. stir-frying, accounted for 35.1% of pork, 25.6% of poultry The results of the present study indicate that, while mean and 47.2% of crustaceans consumed. meat intake among this study population lies within the range described for European populations in the EPIC cohorts, which ranged from 47 to 124 g/day for women, and from 79 to 234 g/day for men, the pattern of consump- Discussion tion differs markedly from populations in Europe (Linseisen There is increasing evidence from basic and epidemiologic et al, 2002), the United States (Byrne et al, 1998) and New research of the potential role of HCAs as a cause of cancer in Zealand (Ferguson, 2002). Compared with the United States populations that consume a Western diet (Layton et al, 1995; (Byrne et al, 1998), Chinese in this population have higher Zimmerli et al, 2001). To our knowledge this is the first study fish consumption (US: 6–15 g/day, current study: 36–44 g/ to describe intake of meat and the cooking methods used in a day), lower beef consumption (US: B20–30 g/day, current Chinese population. The strengths of this study are that data study B1 g/day), and fairly similar consumption of chicken covered both foods consumed at home as well as outside the (US NHS and HPFS data: 21–27 g/day, current study: 13–28 g/ home, and information on cooking method was complete. day). The use of in-person interviews for all 986 subjects also With regard to cooking method, Chinese in Singapore enabled standardized measurement of portion size, and appear to consume a larger proportion of their meat boiled interviewers were able to clarify any uncertainties regarding or steamed, and a smaller proportion fried, roasted or grilled the dishes described by participants. We show that the compared with Western populations. In the EPIC cohort, highest proportion of meat eaten, by weight, comprises fish 28.1% of meat and fish consumption events assessed by 24-h and pork, and identify the specific preparations which recalls involved cooking by frying (including deep-frying employ high-temperature methods and thus represent and meat breaded or battered before frying), 14.2% involved potentially significant sources of dietary HCAs. boiling and 5.1% were classified as stir-frying (Rohrmann Several characteristics of the study population will aid et al, 2002). In a Swedish population survey, 61% of the meat interpretation of the data. The study population, which consumed was fried, 20% baked or roasted, 15% boiled and represented a randomly generated subpopulation of a much 4% grilled (Augustsson et al, 1999). Similarly, an estimated larger population-based cohort of men and women from the 47% of meat in the Swiss diet was fried, 19% grilled and 9% Singapore Chinese Health Study, was very similar to the oven-roasted (Zimmerli et al, 2001). In the US, pan-frying parent cohort in mean age, educational level, marital status, was the most common method for cooking chicken (56%) birthplace and length of residence in Singapore, all of which and fish (54%); whereas between 34% (for steak) and 63% are factors that can affect choice of lifestyle and dietary (for hamburgers) of beef was cooked by broiling/grilling habits. (Bogen & Keating, 2001). Pan-fried meats have been Singapore is a tropical country situated one degree north identified as the largest source of HCA in the US diet, and of the equator. There is therefore no seasonal variation in the chicken the largest source among the various meat types food consumed throughout the year other than a few food (Keating & Bogen, 2001). In contrast, studies in Japan show items associated with festivities, which are usually not that grilled fish is a major dietary source of HCAs, and more consumed in any large amount. Singapore imports almost than 50% of HCA intake is derived from this source

European Journal of Clinical Nutrition Heterocyclic amines in the Chinese diet W-P Koh et al 21 (Kobayashi et al, 2002). Not surprisingly, most of the meat data, surface browning or ‘done-ness’ of meat is widely items for which HCA concentrations have been published accepted as a surrogate for cooking time, weight loss, and in the literature are fried or grilled (Skog et al, 1998). indirectly for HCA content, primarily for beef and bacon The relevance of cooking method to HCA formation lies in (Sinha and Rothman, 1997; Skog et al, 1998). This indicator the maximum internal temperature attained as this para- has a more limited utility where fish, pork and to a lesser meter is thought to combine the processes of heat transfer, extent, chicken, are concerned, since these meats do not water loss and cooking-surface reactions, which in turn share the same variation in ‘done-ness’ as beef and bacon. affect HCA formation (Skog et al, 1995). Significant amounts In conclusion, our findings highlight aspects of the of HCAs begin to form in meats and model systems at Chinese diet which are relevant to HCA formation, and call temperatures of 150oC (302oF) or higher (Bjeldanes et al, for novel approaches to estimating individual exposure to 1982; Knize et al, 1985). Pan-frying, barbecuing and grilling/ dietary HCAs. We believe these results provide a framework broiling are high-temperature cooking methods which heat for the development of a database representative of the food by radiative and conductive processes, and have the Chinese diet that will facilitate further investigation into greatest potential to generate significant amounts of these their association with cancer risk in this, and similar compounds. Oven-roasting and baking are methods that populations. cook meat by indirect convection and at lower temperatures, thus producing low or intermediate levels of HCAs. Boiling, steaming, and stewing without previous browning or brais- Acknowledgements ing are cooking methods at temperatures at or below 100oC This study was supported by the National Medical Research (212oF) and usually produce insignificant amount of HCAs Council in Singapore (NMRC 0521/2001). The Singapore (Bjeldanes et al, 1983; Skog et al, 1998; Bogen and Keating, Chinese Health Study is supported by National Cancer 2001). Institute Grants R01 CA55069, R35 CA53890 and R01 While approximately half of the meat consumed by this CA80205. (P.I. Mimi C. Yu). population, being boiled or steamed, is unlikely to represent a significant source of HCAs, we document a propensity for pan-frying fish, which has been shown to produce high References amounts of HCAs (Wakabayashi et al, 1993). Secondly, Anderson KE, Sinha R, Kulldorff M, Gross M, Lang NP, Barber C, barbecued or roast pork (known locally as ‘char siew’ and Harnack L, DiMagno E, Bliss R & Kadlubar FF (2002): Meat intake ‘siew yoke’) is a significant pork item. This is traditionally and cooking techniques: associations with pancreatic cancer. Mutat. Res. prepared by grilling on a rotating spit over an open fire in a 506–507, 225–231. Archer CL, Morse P, Jones RF, Shirai T, Haas GP & Wang CY (2000): large drum till parts of the surface are crispy or burnt. Carcinogenicity of the N-hydroxy derivative of 2-amino-1-methyl- Our data also show that stir-frying is a common method 6-phenylimidazo[4,5-b]pyridine, 2-amino-3, 8-dimethyl-imida- used to cook pork and chicken, and therefore needs to be zo[4,5-f]quinoxaline and 3, 20-dimethyl-4-aminobiphenyl in the Cancer Lett. explored further. In stir-frying, all ingredients including rat. 155, 55–60. Augustsson K, Skog K, Jagerstad M & Steineck G (1997): Assessment meat, are finely chopped or sliced before cooking, and a large of the human exposure to heterocyclic amines. Carcinogenesis 18, surface area is consequently exposed to heat. The oil is first 1931–1935. heated in a wok or skillet till smoking, the essence of the Augustsson K, Lindblad J, Overvik E & Steineck G (1999): A population-based dietary inventory of cooked meat and assess- technique being that the temperature should be high ment of the daily intake of food mutagens. Food Addit. Contam. 16, enough for the food to be cooked very quickly, often in a 215–225. matter of minutes or even seconds, to retain the moisture Bjeldanes LF, Morris MM, Felton JS, Healy S, Stuermer D, Berry P, and flavor. As the food is fried, it is stirred almost Timourian H & Hatch FT (1982): Mutagens from the cooking of food. II. Survey by Ames/Salmonella test of mutagen formation in continuously so that the hot oil covers every part of the the major protein-rich foods of the American diet. Food Chem. food, hence its name. Recent work suggests that flipping of Toxicol. 20, 357–363. meat patties lowers the surface temperature intermittently Bjeldanes LF, Morris MM, Timourian H & Hatch FT (1983): Effects of and may reduce the amount of HCAs formed (Salmon et al, meat composition and cooking conditions on mutagen formation in fried ground beef. J. Agric. Food Chem. 31, 18–21. 2000), although simulation studies could not fully explain Bogen KT & Keating GA (2001): U.S. dietary exposures to hetero- these reductions based on a thermal (cooking time or cyclic amines. J. Expo. Anal. Environ. Epidemiol. 11, 155–168. temperature-related) effect (Tran et al, 2002). While more Byrne C, Sinha R, Platz EA, Giovannucci E, Colditz GA, Hunter DJ, studies on the mechanism of this effect are needed, it Speizer FE & Willett WC (1998): Predictors of dietary heterocyclic Cancer Epidemiol. remains possible that the effect of very high temperatures amine intake in three prospective cohorts. Biomarkers Prev. 7, 523–529. may in fact be mitigated by the short cooking time and De Stefani E, Ronco A, Mendilaharsu M, Guidobono M & Deneo- constant stirring. Stir-frying remains an important and Pellegrini H (1997): Meat intake, heterocyclic amines, and risk of unique aspect of the Chinese diet and its significance in breast cancer: a case–control study in Uruguay. Cancer Epidemiol. Biomarkers Prev. 6, 573–581. relation to HCA exposure deserves further study. Deneo-Pellegrini H, De Stefani E, Ronco A, Mendilaharsu M & Finally, in epidemiologic studies where HCA exposure Carzoglio JC (1996): Meat consumption and risk of lung cancer; a needs to be estimated from dietary recalls or questionnaire case–control study from Uruguay. Lung Cancer 14, 195–205.

European Journal of Clinical Nutrition Heterocyclic amines in the Chinese diet W-P Koh et al 22 Ferguson LR (2002): Meat consumption, cancer risk and population Monti SM, Ritieni A, Sacchi R, Skog K, Borgen E & Fogliano V (2001): groups within New Zealand. Mutat. Res. 506–507, 215–224. Characterization of phenolic compounds in virgin olive oil and Gerhardsson de Verdier M, Hagman U, Peters RK, Steineck G & their effect on the formation of carcinogenic/mutagenic hetero- Overvik E (1991): Meat, cooking methods and colorectal cancer: a cyclic amines in a model system. J. Agric. Food Chem. 49, case-referent study in Stockholm. Int. J. Cancer 49, 520–525. 3969–3975. Hankin JH, Stram DO, Arakawa K, Park S, Low SH, Lee HP & Yu MC Murtaugh MA, Ma KN, Sweeney C, Caan BJ & Slattery ML (2004): (2001): Singapore Chinese Health Study: development, validation, Meat consumption patterns and preparation, genetic variants of and calibration of the quantitative food frequency questionnaire. metabolic enzymes, and their association with rectal cancer in Nutr. Cancer 39, 187–195. men and women. J. Nutr. 134, 776–784. Hasegawa R, Tanaka H, Tamano S, Shirai T, Nagao M, Sugimura T & Nagao M, Ushijima T, Watanabe N, Okochi E, Ochiai M, Nakagama H Ito N (1994): Synergistic enhancement of small and large & Sugimura T (2002): Studies on mammary carcinogenesis intestinal carcinogenesis by combined treatment of rats with five induced by a heterocyclic amine, 2-amino-1-methyl-6-phenylimi- heterocyclic amines in a medium-term multi-organ bioassay. dazo[4,5-b]pyridine, in mice and rats. Environ. Mol. Mutagen. 39, Carcinogenesis 15, 2567–2573. 158–164. Jagerstad M, Olsson K, Grivas S, Negishi C, Wakabayashi K, Tsuda M, Norrish AE, Ferguson LR, Knize MG, Felton JS, Sharpe SJ & Jackson Sato S & Sugimura T (1984): Formation of 2-amino-3,8-dimethy- RT (1999): Heterocyclic amine content of cooked meat and risk of limidazo[4,5-f]quinoxaline in a model system by heating creati- prostate cancer. J. Natl. Cancer Inst. 91, 2038–2044. nine, glycine and glucose. Mutat. Res. 126, 239–244. Robbana-Barnat S, Rabache M, Rialland E & Fradin J (1996): Kampman E, Slattery ML, Bigler J, Leppert M, Samowitz W, Caan BJ & Heterocyclic amines: occurrence and prevention in cooked food. Potter JD (1999): Meat consumption, genetic susceptibility, and Environ. Health Perspect. 104, 280–288. colon cancer risk: a United States multicenter case–control study. Rohrmann S, Linseisen J, Becker N, Norat T, Sinha R, Skeie G, Lund E, Cancer Epidemiol. Biomarkers Prev. 8, 15–24. Martinez C, Barricarte A, Mattisson I, Berglund G, Welch A, Davey Keating GA, Layton DW & Felton JS (1999): Factors determining G, Overvad K, Tjonneland A, Clavel-Chapelon F, Kesse E, Lotze G, dietary intakes of heterocyclic amines in cooked foods. Mutat. Res. Klipstein-Grobusch K, Vasilopoulou E, Polychronopoulos E, Pala V, 443, 149–156. Celentano E, Bueno-De-Mesquita HB, Peeters PH, Riboli E & Keating GA & Bogen KT (2001): Methods for estimating heterocyclic Slimani N (2002): Cooking of meat and fish in Europe—results amine concentrations in cooked meats in the US diet. Food Chem. from the European Prospective Investigation into Cancer and Toxicol. 39, 29–43. Nutrition (EPIC). Eur. J. Clin. Nutr. 56, 1216–1230. Knekt P, Albanes D, Seppanen R, Aromaa A, Jarvinen R, Hyvonen L, Salmon CP, Knize MG, Panteleakos FN, Wu RW, Nelson DO & Felton Teppo L & Pukkala E (1990): Dietary fat and risk of breast cancer. JS (2000): Minimization of heterocyclic amines and thermal Am. J. Clin. Nutr. 52, 903–908. inactivation of Escherichia coli in fried ground beef. J. Natl. Cancer Knize MG, Andresen BD, Healy SK, Shen NH, Lewis PR, Bjeldanes LF, Inst. 92, 1773–1778. Hatch FT & Felton JS (1985): Effects of temperature, patty Sinha R, Rothman N, Brown ED, Salmon CP, Knize MG, Swanson CA, thickness and fat content on the production of mutagens in fried Rossi SC, Mark SD, Levander OA & Felton JS (1995): High ground beef. Food Chem. Toxicol. 23, 1035–1040. concentrations of the carcinogen 2-amino-1-methyl-6-phenylimi- Knize MG, Sinha R, Rothman N, Brown ED, Salmon CP, Levander dazo- [4,5-b]pyridine (PhIP) occur in chicken but are dependent OA, Cunningham PL & Felton JS (1995): Heterocyclic amine on the cooking method. Cancer Res. 55, 4516–4519. content in fast-food meat products. Food Chem. Toxicol. 33, Sinha R & Rothman N (1997): Exposure assessment of heterocyclic 545–551. amines (HCAs) in epidemiologic studies. Mutat. Res. 376, 195–202. Knize MG, Salmon CP, Pais P & Felton JS (1999): Food heating and Sinha R, Kulldorff M, Curtin J, Brown CC, Alavanja MC & Swanson the formation of heterocyclic aromatic amine and polycyclic CA (1998a): Fried, well-done red meat and risk of lung cancer in aromatic hydrocarbon mutagens/carcinogens. In Impact of Proces- women (United States). Cancer Causes Control 9, 621–630. sing on Food Safety ed. al. Je pp 179–193. New York: Kluwer Sinha R, Knize MG, Salmon CP, Brown ED, Rhodes D, Felton JS, Academic/Plenum Publishers. Levander OA & Rothman N (1998b): Heterocyclic amine content Kobayashi M, Hanaoka T, Nishioka S, Kataoka H & Tsugane S (2002): of pork products cooked by different methods and to varying Estimation of dietary HCA intakes in a large-scale population- degrees of doneness. Food Chem. Toxicol. 36, 289–297. based prospective study in Japan. Mutat. Res. 506–507, 233–241. Sinha R, Gustafson DR, Kulldorff M, Wen WQ, Cerhan JR & Zheng W Lang NP, Butler MA, Massengill J, Lawson M, Stotts RC, Hauer-Jensen (2000): 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, a car- M & Kadlubar FF (1994): Rapid metabolic phenotypes for cinogen in high-temperature-cooked meat, and breast cancer risk. acetyltransferase and cytochrome P4501A2 and putative exposure J. Natl. Cancer Inst. 92, 1352–1354. to food-borne heterocyclic amines increase the risk for colorectal Skog K (1993): Cooking procedures and food mutagens: a literature cancer or polyps. Cancer Epidemiol. Biomarkers Prev. 3, 675–682. review. Food Chem. Toxicol. 31, 655–675. Layton DW, Bogen KT, Knize MG, Hatch FT, Johnson VM & Felton Skog K, Steineck G, Augustsson K & Jagerstad M (1995): Effect of JS (1995): Cancer risk of heterocyclic amines in cooked foods: cooking temperature on the formation of heterocyclic amines in an analysis and implications for research. Carcinogenesis 16, fried meat products and pan residues. Carcinogenesis 16, 861–867. 39–52. Skog KI, Johansson MA & Jagerstad MI (1998): Carcinogenic Linseisen J, Kesse E, Slimani N, Bueno-De-Mesquita HB, Ocke MC, heterocyclic amines in model systems and cooked foods: a review Skeie G, Kumle M, Dorronsoro Iraeta M, Morote Gomez P, Janzon on formation, occurrence and intake. Food Chem. Toxicol. 36, L, Stattin P, Welch AA, Spencer EA, Overvad K, Tjonneland A, 879–896. Clavel-Chapelon F, Miller AB, Klipstein-Grobusch K, Lagiou P, Tran NL, Salmon CP, Knize MG & Colvin ME (2002): Experimental Kalapothaki V, Masala G, Giurdanella MC, Norat T, Riboli E, and simulation studies of heat flow and heterocyclic amine Rohrmann S, Becker N, Sinha R, Lund E, Martinez C, Barricarte A, mutagen/carcinogen formation in pan-fried meat patties. Food Mattisson I, Berglund G, Welch A, Davey G, Lotze G, Vasilopoulou Chem. Toxicol. 40, 673–684. E, Polychronopoulos E, Pala V, Celentano E & Peeters PH (2002): Wakabayashi K, Ushiyama H, Takahashi M, Nukaya H, Kim SB, Meat consumption in the European Prospective Investigation into Hirose M, Ochiai M, Sugimura T & Nagao M (1993): Exposure to Cancer and Nutrition (EPIC) cohorts: results from 24-h dietary heterocyclic amines. Environ. Health Perspect. 99, 129–134. recalls. Cooking of meat and fish in Europe—results from the Wu AH, Shibata D, Yu MC, Lai MY & Ross RK (2001): Dietary European Prospective Investigation into Cancer and Nutrition heterocyclic amines and microsatellite instability in colon adeno- (EPIC). Public Health Nutr. 5, 1243–1258. carcinomas. Carcinogenesis 22, 1681–1684.

European Journal of Clinical Nutrition Heterocyclic amines in the Chinese diet W-P Koh et al 23 Yoshimoto M, Tsutsumi M, Iki K, Sasaki Y, Tsujiuchi T, Sugimura T, meat intake and the risk of breast cancer. J. Natl. Cancer Inst. 90, Wakabayashi K & Konishi Y (1999): Carcinogenicity of hetero- 1724–1729. cyclic amines for the pancreatic duct epithelium in hamsters. Zimmerli B, Rhyn P, Zoller O & Schlatter J (2001): Occurrence of Cancer Lett. 143, 235–239. heterocyclic aromatic amines in the Swiss diet: analytical method, Zheng W, Gustafson DR, Sinha R, Cerhan JR, Moore D, Hong CP, exposure estimation and risk assessment. Food Addit. Contam. 18, Anderson KE, Kushi LH, Sellers TA & Folsom AR (1998): Well-done 533–551.

European Journal of Clinical Nutrition