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I :2. s. . r » .c , z :- ..el.'.:A , . . . . 3 .1 . .. . .. . ~1.. . .9 I. . llBRARY Mid"Han Stale niversity This is to certify that the dissertation entitled Inhibition of heterocyclic aromatic amine formation in fried ground beef patties by organosulfur compounds in garlic presented by Hanseung Shin has been accepted towards fulfillment of the requirements for Ph.D. degreeinmmence Ma jOl' professor J Datesg’ec‘ 6! 2'00 l MS U is an Affirmatiw Action/Equal Opportunity Institution 0-12771 PLACE IN RETURN Box to remove this checkout from your record. To AVOID FINES return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE DATE DUE 6/01 cJCIRC/DateDuepss-p. 15 INHIBITION OF HETEROCYCLIC AROMATIC AMINE FORMATION IN FRIED GROUND BEEF PA'ITIES BY ORGAN OSULFUR COMPOUNDS IN . GARLIC By Hanseung Shin A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of ' DOCTOR OF PHILOSOPHY Department of Food Science and Human Nutrition 2001 ABSTRACT INHIBITION OF HETEROCYCLIC AROMATIC AMINE FORMATION IN FRIED GROUND BEEF PATTIES BY ORGANOSULFUR COMPOUNDS IN ‘ GARLIC By HANSEUNG SHIN The efl‘ect of organosulfur compounds on heterocyclic aromatic amine (HAA) formation and overall mutagenicity in fi'ied beef patties was studied. Organosulfiir compounds (0.67 mmol) were added directly to 100g of ground beef and flied at 225°C for 10 min per side. HAAs were isolated by solid phase extraction and quantitated by high performance liquid chromatography (HPLC). Concentrations of 2-amino-1- methyl-6-phenylimidazo[4,5-b] pyridine (PhIP), the major HAA in muscle foods, were reduced by 81 and 69% by diallyl disulfide (DAD) and dipropyl disulfide (DPD), respectively, while cysteine and cystine were less effective inhibitors. In a second series of experiments, the effect of organosulfur compounds on HAA formation in flied ground beef patties and mutagenicity was evaluated by HPLC analysis and the Ames Salmonella whimurium assay. The greatest inhibition of HAA formation was observed by DAD and DPD, with reductions of 78 and 70%, respectively. The overall mutagenicity of the fried beef patties was reduced 75 and . 65% by DAD and DPD, respectively. The measured mutagenicity in fi'ied beef patties was quite similar to the mutagenicity values calculated from the determined concentrations of HAAs. A series of model system studies were conducted to more completely understand the mechanism by which HAA formation is inhibited by sulfur compounds. The concentrations of PhIP increased 3-4 fold when glucose was added to the model system containing phenylalanine and creatinine. These studies confirmed glucose as an important contributor to HAA formation. Organosulfur compounds and sodium bisulfite effectively inhibited HAA formation in model systems containing A phenylalanine, glucose, and creatinine. However, these compounds had no effect on . HAA formation in the model systems that did not contain glucose. A possible mechanism of HAA inhibition by DAD and DPD in model systems containing glucose could be through their interaction with glucose. A number of sulfur-containing cOmpounds such as tetrahydrothiophene-3 -one (THT) and tetrahydrothiophene (THP) were produced by heating glucose and DAD at 180°C for 30min. However, THT and THP had no effect on HAA formation in the various model systems, regardless of whether glucose was present or not. THT and THP are merely reaction products between glucose and DAD and do not influence HAA formation. While these experiments point to a competitive reaction between organosulfur compounds and amino acids for glucose, the mechanism by which these compounds inhibit HAA formation is still not fully clarified. However, the observation that DAD has no effect on HAA formation in model systems without glucose provides supporting evidence that the interaction of DAD with glucose is a possible key element in its inhibition of HAA formation. It is also apparent that the products of interaction of glucose and DAD are not directly involved in the inhibition process. Dedicate to my parents for helping me find my way in life iv ACKNOWLEDGEMENTS I wish to acknowledge my sincere gratitude to my two advisors, Dr. Gale M. Strasburg and Dr. J. Ian Gray, for their patience, support and encouragement throughout my studies at Michigan State University. Dr. Gray’s enthusiasm for scientific research and motivation for independent thinking will always be an inspiration to me. I also appreciate very much Dr. Gray’s patience in helping me develop my dissertation. Dr. Strasburg has been an excellent advisor and I cannot thank him enough for all the help and guidance he has provided me. Appreciation is also extended to Drs. A.M. Booren and Muraleedharan G. Nair for serving on my guidance committee and all the helpful meetings and suggestions for my research. Sincere thanks and appreciation are given to Dr. Enayat Gomaa for her assistance with the experimental design of the studies and with the extraction and HPLC analyses. I wish to thank my laboratory colleagues, John Rodgers, Aziz Awad, Wen Chiang, Hyojung Yoon, Andrea Molengrafi, and Vareemon Tuntivanich for their invaluable help and encouragement. I also appreciate the helpful suggestions of a number of faculty and stafl‘ members in the Department of Food Science and Human Nutrition. My great appreciation goes to my dear parents Drs. Hyosun Shin and Yangja Yoo who have offered me tremendous support, encouragement, and love throughout my years at Michigan State University. I also thank my two sisters for their concern and support during the completion of this dissertation. Finally, I would like to ofl‘er my special thanks and love to my wife, Sojung Seo, for her support and help along the way. TABLE OF CONTENTS LIST OF TABLES LIST OF FIGURES INTRODUCTION CHAPTER ONE REVIEW OF LITERATURE Heterocyclic aromatic amine in meat systems Quinolines Quinoxalines Pyridines Furopyridines Non-polar heterocyclic aromatic amines Other heterocyclic aromatic mines in food Mutagenicity of heterocyclic aromatic amines Formation of heterocyclic aromatic amine in foods Reactants Effect of cooking time and temperature on HAA formation Chemistry of HAA formation Reduction of heterocyclic aromatic amine formation 1n foods Sugars and other carbohydrates Soy protein concentrate and defatted glandless cottonseed flour Phenolic antioxidants Tea phenolics History and therapeutic effects of garlic The composition of garlic Sulfur compounds in garlic Role of sulfirr compounds in biotransformation of xenobiotics and the inhibition of heterocyclic aromatic vi 19 20 20 21 22 23 24 26 28 29 34 36 36 37 38 39 41 41 amine formation . . . . . . 45 CHAPTER TWO INHIBITION OF HETEROCYCLIC AROMATIC AMINE FORMATION IN FRIED GROUND BEEF PATTIES BY GARLIC AND ITS SULFUR COMPOUNDS ABSTRACT . . . . . . 51 INTRODUCTION I . . . . . . 52 MATERIALS AND METHODS . . . . . . 54 Safety . . . . . . 54 Materials . . . . . . 54 Preparation of ground beef patties . . . . . . 55 Cooking of patties . . . . . . 55 Quantification of HAAs in ground beef patties . . . . . . 56 Statistical analyses . . . . . . 58 RESULTS AND DISCUSSION . . . . . . 58 CHAPTER THREE REDUCTION OF HETEROCYCLIC AROMATIC AMINE FORMATION AND MUTAGENICITY IN FRIED GROUND BEEF PATTIES BY ORGAN OSULFUR COMPOUNDS ABSTRACT . . . . . . 65 INTRODUCTION . . . . . . 66 MATERIALS AND METHODS . . . . . . 68 Safety . . . . . . 68 Materials . . . . . . 68 Preparation of ground beef patties . . . . . . 69 Cooking of patties . . . .' . . 69 Extraction of HAAs from meat samples . . . . . . 69 HPLC analyses . . . . . . 7O Salmonella mutagenicity assay . . . . . . 71 vii Statisticalanalyses ......71 RESULTS AND DISCUSSION . . . . . . 72 Reduction of HAA formation and overall mutagenicity in ground beef patties by organosulfirr compounds . . . . . . 72 Measured and calculated mutagenicity in fried ground beefpatties . . . . . . 74 CHAPTER FOUR A MODEL SYSTEM STUDY OF THE INHIBITIION OF HETEROCYCLIC AROMATIC AMINE FORMATION BY ORGAN OSULFUR COMPOUNDS ABSTRACT . . . . . . 79 INTRODUCTION . . . . . . 80 MATERIALS AND METHODS . . . . . . 82 Safety . . . . . . 82 Materials . . . . . . 82 Effect of organosulfur compounds and sodium bisulfite on HAA formation in model systems . . . . . . 83 Effect of heating on the stability of organosulfur compounds - . . . . . . 84 Effect of heating on sulflrydryl groups . . . . . . 85 Formation of volatile compounds in heated model systems . . . . . . 85 Effect of heating time on HAA formation and inhibition in model systems containing DAD or DPD . . . . . . 86 Efi‘ect of tetrahydrothiphene—3 -one (THT) and tetrahydrothiophene (TI-1P) on HAA formation in various model systems . . . . . . 87 Statistical Analyses . . . . . . 87 RESULTS AND DISCUSSION . . . . . . 88 Efi‘ect of organosulfur compounds and sodium bisulfite on HAA formation in model systems HAA formation . . . . . . 88 Efi‘ect of heating on the stability of organosulfirr compounds . . . . . . . 91 Formation of volatile compounds in heated model system . . . . . . 96 Efi‘ect of heating time on HAA formation and inhibition of HAA formation in model systems containing DAD and DPD . . . . . . 101 viii Efi'ect of tetrahydrothiphene—3-one (THT) and tetrahydrothiophene (THP) on HAA formation in various model systems CHAPTER FIVE SUMMARY AND CONCLUSIONS CHAPTER SIX FUTURE RESEARCH REFERENCES APPENDIX I 103 107 113 115 134 LIST OF TABLES TABLES CHAPTER ONE LITERATURE REVIEW 1. Heterocyclic aromatic amine content of cooked foods 2. Mutagenicity of heterocyclic aromatic amines and typical carcinogens as determined by the Salmoner (whimrm‘um 3. Heterocyclic aromatic amines produced in model systems 4. The general composition of garlic 5. Sulfirrcompoundsinwholeand crushedgarlic cloves CHAPTER TWO 1. Effect of minced garlic cloves on the formation of heterocych aromatic amines in ground beef patties 2. Efl‘ect of variable diallyl disulfide concentrations on the formation of heterocyclic aromatic amines in ground beef patties 3. Efl'ect of various sulfirr compounds on the formation of heterocychc aromatic amines in ground beef patties CHAPTER FOUR 1. Effect of organosulfur compounds and sodium bisulfite on the formation of heterocyclic aromatic amine in model system containing phenylalanine, creatinine, and glucose 2. Effect of organosulfirr compounds and sodium bisulfite on the formation of heterocyclic aromatic amine in model system containing phenylalanine and creatinine 25 27 42 46 6O 63 89 92 3.Compoundstentativelyidentifiedonheating g1ucoseat180°Cfor30min 4. Compounds tentatively identified on heating diallyldisulfide at 180°C for 30 min 5. Compounds tentatively identified on heating glucose and diallyldisulfide at 180°C for 30 min 6. Efi‘ect of tetrahydrothiophene-3 -one (THT) and tetrahydrothiophene (THP) on formation of heterocychc aromatic amine in a model system containing phenylalanine, creatinine, and glucose 7. Efi’ect of tetrahydrothiophene-3 -one (THT) and tetrahydrothiophene (THP) on formation of heterocyclic aromatic amine in model system containing phenylalanine and creatinine 8. Effect of tetrahydro thiophene-3 -one (THT) and tetrahydrothiophene (THP) on formation of heterocyclic aromatic amine in a model system containing glycine, creatinine, and glucose 97 98 100 104 105 106 LIST OF FIGURES FIGURES Page CHAPTER ONE LITERATURE REVIEW 1. Chemical structures of some HAAs in cooked foods . . . . . . 6 2. Initial steps ofthe Maillard reaction . . . . . . 3] 3. A suggested pathway of browning in Maillard reaction through a free radical . . . . . . 33 4. Theoretical reaction pathway for formation of IQ and IQx compounds . . . . . . 35 5. Sulfur content of common fruits and vegetables . . . . . . 42 6. Formationofallicinbytheaction ofalliinaseand transformation of the principal thiosulfinates of crushed garlic . . . . . . 47 CHAPTER TWO CHAPTER THREE 1. The effect of organosulfur compounds on the mutagenicity of ground beef patties as determined by AmesSOphimuriumTA98assay . . . . . . 75 2. Plot of mutagenic activity quantitated by S. whimurium TA98 assay and mutagenic activity calculated fi'om the heterocyclic aromatic amine concentratons in fiied beef patties as determined by HPLCanalyses ......78 CHAPTER FOUR 1. Effect of heating at 180 °C for 30 min on the concentrations of organosulfur compounds in a model system containing phenylalanine, creatinine, and glucose . . . . . . 94 2. Sulthydryl content on heating DAD and DPD at xii 180 °C for 30min 95 3. Inhibition of total HAA formation by DAD and DPD in a model system containing phenylalanine, creatinine, and glucose heated at 180 °C for 30 min . . . . 102 xiii INTRODUCTION Heterocyclic aromatic amines (HAAs) are commonly found in meat and fish products cooked at temperatures greater than 150 °C. These compounds have been classified into two categories, pyrolytic mutagens and thermic mutagens, based on their temperatures of formation. Pyrolytic mutagens are formed when proteins and/or amino acids are heated to high temperatures (>300 °C) and are characterized by a pyridine ring with an amino group attached (Skog, 1993; Wakabayashi and Sugimura, 1998). Thermic mutagens are formed at lower temperatures (<3 00 °C), and several have been identified in cooked muscle foods. These compounds, also called aminoirnidazoazaarenes, can be broken down into four major categories: quinolines, quinoxalines, pyridines, and filropyridines. The most commonly found HAAs in foods are IQ (2-amino-3-methy1imidazo[4,5-j]-quinoline); MeIQ (2-arnino- 3,4dimethylimidazo[4,Sfl-quinoline); MeIQx (2-amino-3,8 dimethylimidazo [4,5-j]- quinoxaline); 4,8 DiMeIQx (2-amino-3,4,8 trimethylimidazo[4,5-j]-quinoxaline); and PhIP (2—amino-1-methyl-6-phenylimidazo [4,5-b]-pyridine) (Knize et al., 1999; Skog, 1993; Wakabayashi and Sugimura, 1998). Many of the HAAs isolated from foods have been shown to be mutagenic by the Ames Salmonella {whimurium mutagenicity assay (F elton et al., 1997) and by mammalian cell culture studies such as those employing Chinese hamster ovarian cells (Holrne et al., 1989). Mutagenicity varies widely among individual HAAs, and has been reported to be as high as 661,000 revertants/ug toward S. typhimurium TA98. Aflatoxin Bl, a well-documented carcinogen, causes only 6,000 reverents/ug under the same assay conditions. It has been reported that HAAs, when added to diets, will ‘ produce carcinogenic lesions in mice and rats (Esumi et al., 1989). Because HAAs are found in a variety of cooked foods which constitute a major part of the diet of the US. population, they are considered to be potential risk factors for human health (Hirose et al., 1999). The precursors of HAAs in cooked meat products are thought to be creatine/creatinine, amino acids and sugars (Jagerstad et al., 1983a). It has been suggested that HAA formation follows the Maillard reaction through the generation of vinylpyrazine, vinylpyridine and aldyhydes (lagerstad et al., 1983b). Factors influencing HAA formation include the temperature, time, and method of cooking, as well as the concentrations of precursors present in the food (Knize et al., 1994; Skog et al., 1992). Several approaches to decreasing HAA formation in food systems have been suggested. Concentrations of HAA precursors in meat patties (creatine, amino acids and sugar) are reduced by microwave pretreatment of the patties before flying (F elton et al., 1992). Addition of glucose or lactose at levels. ranging from 2 to 4 percent will reduce the overall mutagenicity of cooked ground meat (Skog et al., 1992). Food ingredients, such as vitamin E and tea phenolic antioxidant compounds, will reduce HAA formation in meat (Balogh et al., 2000; Weisburger et al., 1994). Soy protein concentrates (Wang et al., 1982) and the marinating of meats before cooking (Salmon et al., 1997) will also inhibit HAA formation. Sulfur compounds have been reported to provide various health-promoting benefits including antiplatelet activity (Bordia et al., 1998), antiproliferative activity against human colon tumor cells (Knowles and Milner, 1998), and the lowering of plasma and liver cholesterol levels (Omkumar et al., 1993). Several studies have revealed the ability of selected classes of sulfur compounds to inhibit HAA formation in meats and model food systems. Addition of sulfur compounds to an aqueous pork extract reduced the formation of Maillard reaction products with a concomitant decrease in mutagenicity (Tsai et al., 1996). Trompeta and O’Brien (1998) demonstrated that sulfirr compounds such as glutathione, L-cysteine, L-cystine, and deoxyalliin inhibited the formation of HAAs in model systems containing glucose, glycine, and creatinine. Furthermore, the addition of onion juice to ground beef reduced HAA formation (Kato et al., 1998). The mechanism(s) by which sulfur compounds inhibit HAA formation during the cooking of meats has/have not been fully investigated. Several investigators have proposed possible mechanisms including the inhibition of the Maillard reaction and the suppression of fiee radical formation by thiol trapping (Friedman and Molnar-Perl, 1990; Tsai et al., 1996). i This study is based on the hypothesis that sulfur compounds, when added to model systems containing creatinine, amino acids and glucose, and ground beef patties, will inhibit HAA formation through selected competitive reactions. This hypothesis is based on the premise that the Maillard reaction plays an integral role in the formation of HAAs. To test this hypothesis, a number of objectives were developed to not only confirm the inhibitory role of selected sulfur compounds in meat and model systems, but also to gather data that will lead to a fuller understanding of HAA inhibition by sulfur compounds. Specific objectives of the study are as follows: 1. To evaluate the effect of selected organosulfur compounds present in garlic on HAA formation in fn'ed ground beef patties. 2. To investigate the relationship between the reduction of specific HAA compounds and overall mutagenicity of the cooked meat system as determined by the Ames Salmonella typhimurium assay. 3. To conduct selected experiments that will lead to a more complete understanding of the inhibition of HAA formation by sulfur compounds. CHAPTER ONE LITERATURE REVIEW Heterocyclic Aromatic Amines in Meat Systems Heterocyclic aromatic amines (HAAs) have been classified into two categories based on their temperatures of formation: pyrolytic mutagens and thermic mutagens. Pyrolytic mutagens are formed when proteins or amino acids are heated to temperatures in excess of 300°C and are characterized by a pyridine ring with an amino group attached (Skog, 1993). Thermic mutagens are formed at lower temperatures (<300 °C), and several have been identified in cooked meat and fish. These mutagens, also called aminoirnidaaoazaarenes, can be broken down into four categories: quinolines, quinoxalines, pyridines, and furopyridines. Several have been isolated and identified in cooked meat, fish, and food-grade beef extracts (Table 1). The most commonly formed HAAs in meat products are IQ (2-amino-3-methylimidazo[4,Sfl-quinoline); MeIQ (2- amino-3,4dimethylimidazo[4,5;/]-quinoline); MeIQx (2-amino-3,8 dimethylimidazo [4,5-fl-quinoxaline); 4,8 DiMeIQx (2-amino-3,4,8 trimethylimidazo[4,5—j]- quinoxaline); and PhIP (2-amino-1-methyl-6-phenylimidazo [4,5-b]-pyridine) (Skog, 1993). Their structures are shown in Figure 1. uinolines Two imidazoquinoline compounds, IQ and MeIQ, were first identified by Kasai et al. (1980) in broiled sun-dried sardines. These compounds have been subsequently NHz N7< N—CH3 / O \ N IQ NH: N/ N—CH3 H3C N g \< \ N MeIQx PhIP NH; NA N . IQ! ’ Ha Hz N / N— c143 H3C N / CHs \ N 4,8-DiMeIQx Figure 1. Chemical structures of some HAAs found in cooked foods (Skog, 1993). Table 1. Heterocychc aromatic amine content of cooked foods. Foodtype Mutagen Amount] Temp2 Time Weight‘ Ree BEEF Fried Mele 0.5 O 43 4,8-DiMeIQx 3.9 275 15 0 38 8-MeIQx 12.3 200 15 0 38 8-MeIQx 4.0 150 6 0 25 IQ 1.9 200 15 0 . 38 Heated Bouillon 4,8-DiMeIQx 0.3 0 33 8-MeIQx 0.6 0 33 PhIP 0.3 0 33 Boiled Extract 4,8-DiMe1Qx 28.0 1 14 4,8-DiMeIQx 0.0-3.7 1 39 4,8-DiMeIQx 0.0-4.4 1 9 4,8-DiMeIQx 0.0 l 33 4,8-DiMeIQx 2.5-4.9 1 10 7,8-DiMeIQx 0.0 l 39 8-MeIQx 28.0 1 14 8-MeIQx 3.1 1 41 8-MeIQx 20.5 1 38 8-MeIQx 8.5-30.0 1 9 8-MeIQx 23.0—69.0 1 40 8-MeIQx 0.0-44.0 1 10 8-MeIQx 0.6 l 33 8-MeIQx 3.1 1 43 8-MeIQx 1 1.7-36.4 1 39 AorC 0.0 1 10 1Q 0.0 1 41 IQ 0.0 1 14 IQ 0.5 1 36 IQ 4.8-6.2 l 40 IQ 0.0-6.2 1 39 IQ 0.0-8.0 1 9 PhIP 3.62 l 15 PhIP 0.0 l 9 PhIP 0.0 1 19 I '31 1.1-5 " 1. Food type Mutagen Amount‘ Temp2 T111163 Weight“ Ref6 PhIP 0.0 l 33 4'-OH-PhIP 2 l .0 l 5 60 l 27 Heated Extract 4—CHzOH-8-Mcx 6 .2 12 l 60 l 24 4-CHzOH-8-Mex 6.7 160 300 1 24 4-CH20H-8-Mex 7.2 200 300 l 24 Flavor 4,8-DiMeIQx 0.0 l 16 4,8-DiMeIQx 0.0 1 16 Mele 0.0-12.5 1 16 Mele 0.0-4.4 1 l6 Roasted 4,8-DiMe1Qx 0.0 1 16 Mele 0.0-4.4 l 16 Grilled 4,8-DiMeIQx 0.0 l 16 Mele 0.0 1 16 GROUND BEEF Charbroiled 4,8-DiMeIQx 0.2 6 1 l7 4,8-DiMeIQx 0.1 10 1 17 MeIQ 0.0 6 1 17 MeIQ 0.4 10 1 17 MeIQx 1.0 6 1 17 Mele 0.4 10 1 17 Mele 0.2-1.8 1 47 IQ 0.0 6 1 17 IQ 0.1 10 1 17 PhIP 0.0 6 l 17 PhD3 5.2-18.4 1 47 Broiled 1Q 0.5 1 44 Grilled AaC 0.0 270 3 l 12 AOLC 0.0 270 5 l 12 AOLC 0.0 270 7 1 12 4,8-DiMeIQx 0.0 270 3 l 12 4,8-DiMeIQx 0.0 270 5 l 12 4,8-DiMeIQx 0.0 270 7 1 12 Mele 0.8 270 3 1 12 Mele 2.0 270 5 1 12 Mele 1.3-4.6 1 47 PhIP 0.7 270 3 l 12 PhIP 1.4-4.8 270 5 l 12 Pth 16.8 1 47 Food type Mutagen Amount' 1'61an Time3 Weight‘ Res PhIP 0.0 270 7 1 12 Fried Aoc 21.0 277 6 1 37 DiMeIQx 4.5 277 6 1 37 DiMeIQx 4.8 225 10 1 49 4,8-DiMeIQx 0.5 300 6 0 7 4,8-DiMeIQx 3.9 275 15 0 39 4,8-DiMele 0.0 275 5 0 39 4,8-DiMeIQx 0.0 275 10 0 39 4,8-DiMeIQx 0.5-1.2 200 2 32 4,8-DiMeIQx 0.12 0 35 4,8-WCIQX 0.3 0 25 4,8-DiMeIQx ‘ 0.0-0.28 2 5 4,8—DiMeIQx 0.54 250 12 1 25 4,8-DiMeIQx 0.1 150 6 0 26 4,8-DiMeIQx 0.0 150 2 0 26 4,8-DiMeIQx 0.0 150 4 0 26 4,8-DiMeIQx 0.7 150 10 0 26 4,8-DiMeIQx 0.0 190 2 0 26 4,8-DiMeIQx 0.10 190 4 0 26 4,8-DiMeIQx 0.55 190 6 0 26 4,8-DiMeIQx 2.6 190 10 0 26 4,8-DiMeIQx 0.15 230 4 0 26 4,8-DiMeIQx 0.25 230 6 0- 26 4,8-DiMele 9.35 230 10 0 26 4,8-DiMcIQx . 0.7 225 6 1 29 4.8-DiMeIQx 3.1 1 25 7,8-DiMeIQx 0.0 275 5 0 39 7,8-DiMeIQx 0.0 275 10 0 39 7,8-DiMeIQx 0.7 275 15 0 39 4-MeIQ 0.1 300 6 0 7 Mele 1.0 250 6 1 6 Mele 16.4 277 6 1 37 Mele 1.0 300 5.5 0 4 Mele 0.0 0 19 Mele 0.0-0.68 2 5 Mele 0.3 0 43 Mele 1.3-2.4 200 2 32 Mele 5.8 225 10 1 49 Food type Mutagen Amount' Teran Time3 Weight“ Ref° Mele 2.7 275 5 0 39 Mele 4.2 275 10 0 39 Mele 12.3 275 15 0 39 Mele 0.5-1.5 0 40 Mele 13-15 1 47 8-MeIQx 0.1 0 6 8-MeIQx 0.45 190 0 14 8-MeIQx 1.1 250 10 1 9 8-MeIQx 1.0 300 6 0 7 8-MeIQx 0.64 0 35 8-MeIQx 0.3 1 25 8-MeIQx 2.95 250 6 1 25 8-MeIQx 0.0 150 2 0 26 8-MeIQx 0.0 150 4 0 26 8-MeIQx 0.15 150 6 0 26 8-MeIQx 2.7 150 10 0 26 8-MeIQx 0.1 190 2 0 26 8-MeIQx 0.25 190 4 0 26 8-MeIQx 1.3 190 6 0 26 8-MeIQx 5.1 190 10 0 26 8-Me1Qx 0.0 230 2 0 26 8-MeIQx 0.4 230 4 0 26 8-MeIQx 1.1 230 6 0 26 8-MeIQx 8.0 230 10 0 26 8-MeIQx 2.2 225 6 1 29 8-Mele 10.8 1 25 IQ 05-200 240 5 0 1 IQ 0.02 250 6 1 6 IQ 0.5 2 44 IQ 0.0 192 0 18 IQ 0.3 275 5 0 39 IQ 0.3 275 10 0 39 IQ 1.9 275 15 0 39 IQ 0.02 300 5.5 1 4 IQ 0.0 150 4 0 26 IQ 0.1 150 6 0 26 IQ 1.5 150 10 0 26 IQ 0.1 190 2 0 26 IQ 0.1 190 4 0 26 IQ‘ 5.3 225 10 l 49 10 Food type Mutagen Amount' Temp Time’ Weight“ Res IQ 0.45 190 6 0 26 1Q 0.82 190 10 0 26 IQ 0.0 230 2 0 26 IQ 0.15 . 230 4 0 26 IQ 0.25 230 6 0 26 IQ 1.8 230 10 0 26 IQ 0.0 250 10 1 9 PhIP 15.0 300 5.5 l 4 PhIP 67.5 277 6 l 37 PhIP 1.2 250 10 1 9 PhIP 5.0 l 25 PhIP 0.56 l 15 PhIP 0.0 150 4 0 26 PhIP 0.25 150 6 0 26 PhIP 0.9 150 10 0 26 PhIP 0.15 190 4 0 26 PhIP 1.9 190 6 0 26 PhIP 6.0 190 10 0 26 PhIP 0.55 230 2 0 26 PhIP 1.35 230 4 0 26 PhIP 4.1 230 6 0 26 PhIP 21.5 230 10 O 26 PhIP 16.4 225 6 1 29 PhIP 21.8 1 25 PhIP 1.9-2.6 l 47 PhIP 31 225 10 l 49 TMIP 0.5 300 6 0 7 Trp-P-l 0.19 0 35 Trp-P-2 0.0 200 0 31 Trp-P—2 0.21 0 35 BEEF STEAK Broiled or Fried 4,8-DiMeIQx 1.3 190 3 1 10 4,8-DiMeIQx 2.0 190 6.5 1 10 4,8-DiMeIQx 0. l 225 6 1 33 8-MeIQx 2.11 0 35 8-MeIQx 5.1 190 3 1 10 8-MeIQx 8.3 190 6.5 1 10 8-MeIQx 0.5 225 6 1 33 Mele 1.7-2.4 l 47 PhIP 6.8-10.0 1 47 DiMeIQx 0.1-0.4 1 47 11 :1'3'7’ Food type Mutagen Amountl Temp2 Time3 Weight4 Ret‘ Aoc 1.2 0 35 AorC 3.2 190 3 l 10 AaC 8.9 190 6.5 1 10 Glu-P-l 0.0 0 18 IQ 0.19 0 35 PhIP l 5.7 l 15 PhIP 23.5 190 3 1 10 PhIP 48. 5 190 6.5 l 10 PhIP 0.6 22 5 6 1 3 3 Trp—P-l 53.0 0 45 Trp—P-l 0.21 0 35 Trp-P-Z 0.25 0 35 Grilled MeIQx 0.2-2.7 160 48 PhIP 25-300 160 48 Charbroiled MeIQx 1.1-1.6 160 48 PhIP 57-15 160 48 Grilled Bonito 4,8-DiMe1Qx 5.4 220 15 1 22 8-MeIQx 5.2 220 15 1 22 CHICKEN Charbroiled 4,8-DiMeIQx 0.1 l 33' 8-MeIQx 0.3 1 33 Broiled 4,8-DiMeIQx 0.81 1 35 Mele 2. l 44 8-MeIQx 2. 33 1 35 AaC . 0.2 l l 35 PhIP 38. l l 15 Trp—P-l 0.12 1 35 Trp-P2 0.18 1 35 Fried Trp-P-l 0.0 300 6 0 8 Consomme, Heated 4,8-DiMeIQx 0.0 0 33 8-MeIQx 0.1 0 33 PhIP 0.0 0 3 3 EEL Fried, Canned 7,8-DiMeIQx 5.3 180 4 1 23 8-MeIQx 1.1 180 4 1 28 12 Food type Mutagen Amountl Tempz T111193 Weight“ Ref‘ Boiled or . 4,8-DiMeIQx 0.0 160 2.5 1 17 Smoked MeIQ 0.0 160 2.5 1 17 Mele 0.6 160 2.5 l 17 IQ 0.3 160 2.5 1 l7 PhIP 0.0 160 2.5 l 17 FISH Smoked Flounder 4,8-DiMeIQx 0.6 1 17 MelQ 0.3 l 17 Mele 0.0-2.9 1 l7 IQ 0.7 1 l7 PhIP 0.0 1 l7 Fried Herring 4,8-DiMeIQx 0.3 l 17 MeIQ 0.1 1 17 Mele 0.6 1 l7 IQ 0.2 1 l7 PhIP 0.0 1 l7 Fried Pollack ‘ 4-MeIQ 0.03 260 8 1 46 4,8-DiMeIQx 0. l 260 8 1 46 8-MeIQx 6.44 260 8 l 46 IQ 0.16 260 8 1 46 , PhIP 69.2 260 8 l 46 Baked Salmon 8-MeIQx 0.0 200 20 1 1 1 8-MeIQx 4.6 200 30 l l 1 8-MeIQx 3.1 200 40 l 1 1 AaC 0.0 200 20 1 1 1 AaC 0.0 200 30 1 1 1 AaC 0.0 200 40 l 1 l PhIP 0.0 200 20 1 l l PhIP 18.0 200 30 1 ' 1 1 PhIP 5.9 200 40 l 1 1 Broiled Salmon Flesh 4-MeIQ 0.6-2.8 l 44 Skin 4-MeIQ 1.1-1.7 1 44 4—MeIQ 0.1-0.9 1 3 MeIQ 1.4-5 .0 l l 1 Flesh IQ 0.3-1.8 l 44 13 Food type Mutagen Amount1 Teran Time3 Weight4 Ref6 Broiled Salmon Sla'n IQ 1.1-1.7 1 44 Broiled Salmon IQ 0.2-0.4 l 3 PhIP 1.7-23 .0 1 1 l Charbroiled Salmon 8-MeIQx 0.0 270 4 l l l 8-MeIQx 0.0 270 6 1 1 l 8-MeIQx 0.0 270 9 l 1 1 8-MeIQx 0.0 270 12 l 11 AaC 2.8 270 4 1 1 l AaC 6.9 270 6 1 l l AaC 73.0 270 9 1 l l AaC 109.0 270 12 1 1 l PhIP 2.0 270 4 l 1 l PhIP 6.2 270 6 l l l PhIP 69.0 270 9 1 1 l PhIP 73 .0 270 12 l l l Cooked Salmon 4,8-DiMele 0.2 150 9 l 17 MeIQ 1.0-1.6 150 9 1 17 Mele 0.6 150 9 l 17 IQ 0.6 150 9 l 17 PhIP 2.7-3.3 150 9 l 17 Fried Salmon 8-MeIQx 1.4 200 3 l l l 8-MeIQx 5 .0 200 6 1 l 1 8-MeIQx 4.7 200 9 1 1 l 8-MeIQx 3.7 200 12 1 1 l AaC 0.0 200 3 l l 1 AorC 4.6 200 6 1 1 1 AaC 8.0 . 200 9 1 ll AaC 9.0 200 12 1 1 l PhIP 1.7 200 3 l l 1 PhIP 23 .0 200 6 l 1 l PhIP 14.0 200 9 1 1 l PhIP 17.0 200 12 l l 1 Smoked Salmon 4,8-DiMeIQx 0.0 0 17 MeIQ 0.0 0 17 Mele 1.2-1.4 0 17 IO 0.3 0 17 PhIP 0.0 0 l7 Broiled Sardine 4-Me1Q 16.6 1 45 14 Foodtype Mutagen Amountl Temp2 Time3 Weight‘ Ref‘ Broiled Sardine 4-MeIQ 20.0 0 20 8-MeIQx 0.0 2 45 Glu-P-l 0.0 l 45 IQ 20.0 0 19 IQ 4.9 1 44 IQ 20.0 0 20 Phe-P-l 8.6 1 45 Trp-P-l 13.3 1 45 Trp-P-2 13.1 1 45 UNSPECIFIED Fried Trp-P-2 0.0 200 1 3 1 Heated 4,8-DiMeIQx 5 .4 l 23 Mele 5 .2 1 23 Smoked, Dried 4,8-DiMeIQx 0.08 1 2 1 Mele 0.8 1 21 LAMB Broiled 4,8-DiMele 0.67 1 35 8-MeIQx 1.01 1 35 AaC 2.5 l 35 AMaC 0. 19 l 3 5 PhIP 42.5 1 15 Trp-P-Z 0.15 l 35 Fried Meatballs 4,8-DiMeIQx 0.2 l 17 MeIQ 0.3 1 17 Mele 0.7 l 17 IQ 0.2 l 17 PhIP 0.6 1 17 Boiled Meat Extract 4,8-DiMeIQx 2 .9-3 .6 1 34 8-MeIQx 6.2-28.3 1 34 IQ 1.9-4.8 1 34 PORK Charbroiled 4,8-DiMeIQx 0.1 0 33 8-MeIQx 0.4 0 33 PhIP 4.2 0 33 Fried Trp-P-l 0.0 300 0 8 15 Food type Mutagen Amount 1 Temp2 Time3 Weight4 Ref5 Fried Bacon MeIQx 0.9-18.0 12-16 1 12 4,8-DiMeIQx 0.0-1. 12-16 1 12 PhIP 0.0-53.0 12-16 1 12 Mele 0.4-4.3 160 l 48 PhIP 0.7-4.8 160 1 48 Moderate 4,8-DiMeIQx 1.7-5.1 150 2.5 1 l7 Well-done 4,8-DiMeIQx 1.0 150 5 l 17 Well-done MeIQ 1.4-2.0 150 5 1 17 Moderate MeIQx 0.0-5.8 150 2.5 l 17 Well-done MeIQx 1.4-3.6 150 5 l 17 Moderate IQ 2.3-5.3 150 2.5 l 17 Well-done IQ 95-115 .150 5 1 17 Moderate PhIP 0.2 150 2.5 l 17 Well-done PhIP 1.0 150 5 1 l7 Broiled Bacon MeIQx 1.5-4.0 160 1 48 PhIP 1.4-30.3 160 l 48 Mcrowaved Bacon MeIQx 0.4-1.5 l 47 PhIP 3.1 l 47 Fried Bacon Fatty 4,8-DiMeIQx 0.3 225 6 1 33 8-McIQx 1.2 225 6 1 33 PhIP 2.7 225 6 l 33 Fried Bacon Lean 4,8-DiMeIQx 0.2 225 6 r 33 . 8-MeIQ 0.9 225 6 1 33 PhIP 1.6 225 6 1 33 Fried Ground Pork 4,8-DiMeIQx 0.6 250 5 0 42 4,8-DiMele 0.24 180 l 2 4-MeIQ 0.02 250 5 0 42 4-MeIQx 0.1 250 5 0 42 4-MeIQx 1.4 250 0 42 4-MCIQx 0.4 180 1 2 IQ 0.04 250 5 0 42 IQ 0.01 180 1 2 Pb]? 4.5 250 5 0 42 PhIP 1.7 180 l 2 PhIP 0.0 0 13 16 [i " —‘._.I'-.l‘1 i v! u Food type Mutagen Amount1 Temp2 Time3 Weight4 Ref“ Fried Ground Pork 4,8-DiMeIQx 0.9 250 1 2 8-MeIQx 1.5 250 1 2 1Q 0.04 250 1 2 PhIP 10.0 250 1 2 ' Fried Pork Sausage 4,8-DiMeIQx 0.2 160 6 l 17 MeIQ 0.2 160 6 l 17 Mele 0.7 160 6 1 l7 IQ 0.1 160 6 l 17 PhIP 0.1 160 6 1 17 Trp-P-l 0.0 300 6 0 8 Ach = 2-amino-9H—pyrido[2,3-b]indole; AMorC = 2-amino-3 -methy1-9H-pyrido[2,3- b]indole; 4—OH—PhIP = 2-amino-l-methy1-6-(4-hydroxypheny1)imidazo[4,5-b]pyridine; 4-CH20H-8-Mex = 2-arninoA—hydroxy-methyl-3,8-dimethy1imdazo[4,S-f] quinoxaline; Trp-P-l = 3-arnino—L4-dimethyl-5H-pyrido[4,3-b]indole; Trp-P-2 = 3-amino- 1-methy1-5H—pyrido[4,3-b]indole; Glu-P-l = 2-amino-6- methyldipyrido[l,2‘-a:3’,2’-flimidazole; Glu-P-Z = 2-aminodipyrido[l,2-a:3’,2’-d] imidazole. 1 Amount of mutagen formed (ng/g) 2 Temperature of frying (°C) 3 Cooking time (minutes per side), except for sausage (total frying time) '4 Basis for heterocyclic aromatic amine concentration. 0 = cooked weight of food, 1 = uncooked weight, 2 = unspecified 5 References: 1- Barnes et al., 1983 2- Dragsted, 1992 3- Edmonds et al., 1986 4- Felton et al., 1986a 5- Felton et al., 1992 6- Felton et al., 1984 7- Felton et al., 1986b 8- Felton et al., 1997 9- Gross et al., 1989 10- Gross, 1990 11- Gross and Grater, 1992 12-Gross et al., 1993 13- Gry et al., 1986 14- Hargraves and Pariza, 1983 15- Hayatsu et al., 1991 16- Jackson et al., 1994 17 17- Johansson and lagerstad, 1994 18- Kasai et al., 1981b 19- Kasai et al., 1981a 20- Kasai et al., 1980 21- Kato et al., 1986 22- Kikugawa et al., 1986 23- Kikugawa and Kate, 1987 24- Kim et al., 1994 25- Knize et al., 1990 26- Knize et al., 1994 27- Kurosaka et al., 1992 28- Lee and Tsai, 1991 29- Lynch et al., 1992 31- Murray et al., 1987 32- Murray et al., 1988 33- Murray et al., 1993 34- Schuirmann and Eichner, 1991 35- Sugimura et al., 1988 36- Taylor et al., 1985 37- Thiebaud et al., 1994 38— Turesky et al., 1983 39- Turesky et al., 1988 40- Turesky et al., 1989 41- Takahashi et al., 1985 42- Vahl et al., 1988 43- Wakabayashi et al., 1986 44- Yamaizumi et al., 1986 45- Yamaizumi et al., 1980 46- Zhang et al., 1988 47- Sinha et al., 1998 48- Knize et al., 1998 49- Balogh et al., 2000 identified in fiied ground beef (Barnes et al., 1983; Felton et al., 1984; Felton et al., 1986b; Turesky et al., 1983), boiled beef extract (Gross et al., 1989; Hargraves and Pariza et al., 1983), charbroiled ground beef (Johansson and lagerstad, 1994), broiled ground beef (Y amaizurni et al., 1986), broiled or fi'ied steak (Sugimura et al., 1988), smoked, fiied, and broiled fish (Johansson and lagerstad, 1994; Yamaizumi et al., 1986; Zhang et al., 1988), fiied meat balls (Johansson and Jagerstad, 1994), and fi'ied ground pork (Vahl et al., 1988). The presence of IQ in fiied ground beef has been reported at 18 ...l 1“ various concentrations ranging from 0 to 20 ng/g (Barnes et al., 1983; Felton et al., 1984; Turesky et al., 1988). However, MeIQ was found in relatively smaller concentrations (Felton et al., 1986a; Gross et al., 1993; Yamaizumi et al., 1986). 92m gxalines The quinoxaline, Mele, was first isolated from flied ground beef, followed by 2-amino—3,7,8-trimethyl imidazo[4,5-fl-quinoxaline (7,8-DiMeIQx) (Negishi et al., 1984), and 4,8-DiMeIQx (Grivas et al., 1985). Mele was identified when a model system containing creatinine, glucose and glycine was heated for two hours at 128 °C. Overvik et a1. (1989) showed that model systems containing creatinine, threonine, serine, or alanine produced MeIQx. 7,8-DiMeIQx has been found in boiled beef extract (Turesky et al., 1988), flied ground beef (Felton et al., 1986b; Turesky et al., 1988), and roasted, flied, and canned» eel (Lee and Tsai, 1991). 4,8-DiMeIQx has been isolated from flied, charbroiled, and grilled ground beef (Felton et al., 1986a, 1992; Grivas et al., 1985; Murray et al., 1988; Sugimura et al., 1988; Turesky et al., 1988), broiled and roasted beef extracts (Gross et al., 1989, 1990; Hargraves and Pariza, 1983; Murray et al., 1993; Turesky et al., 1988), grilled bonito (Kikugawa et al., 1986), charbroiled and broiled chicken (Murray et al., 1993; Sugimura et al., 1988), boiled, smoked, and flied sausage (Johansson and lagerstad, 1994), smoked and flied fishes (Johansson and lagerstad,‘1994; Zhang et al., 1988), broiled lamb (Sugimura et al., 1988), flied meatballs (Johansson and lagerstad, 1994), charbroiled pork (Murray et al., 1993 ), and flied bacon (Murray et al., 1993). 19 Pyridines The most abundant HAA in cooked meat is Phfl’ (Felton et al., 1986a; Gry et al., 1986; Hayatsu et al., 1991; Skog et al., 1997; Vahl et al., 1988). It was first isolated flom the crust of flied ground beef (Felton et al., 1986b). Phenylalanine and glucose have been reported to be its precursors (Felton and Knize, 1990; Manabe et al., 1992; Overvik et al., 1989; Shioya et al., 1987; Skog and lagerstad, 1991). There is some evidence that leucine may also be involved in PhiP formation (Overvik et al., 1989). , Skog and Jagerstad (1990, 1991) demonstrated the effect of glucose on the formation of PhIP by heating a mixture of creatine, phenylalanine and glucose in diethylene glycol and water. In addition, Mele and 4,8-DiMeIQx were also formed. Heating the model system without glucose produced PhIP as a single mutagen, but in a relatively smaller amount. Recently, Arvidsson et a1. (1999) reported that PhIP and other polar HAAs were formed in heated mixtures containing creatinine, glucose and amino acids in proportions similar to those in bovine meat but at higher concentrations. Furonxriging The forementioned HAAs account for 30-7 5% of the total mutagenicity in various cooked foods (Stavric, 1994). Other less-well characterized mutagenic HAAs are present in cooked meat products (Felton et al., 1984, 1986b; Skog et al., 1998; Zhang et al., 1988). A methylimidazo—furopyridine (MeIFP) containing oxygen, with a molecular weight of 202, has been identified in flied minced beef containing milk and creatine (Knize el al., 1990). These investigators suggested that this mutagen is related to the food mutagen with a molecular weight of 216, an arnino- 20 dimethylimidazofirropyridine. Such HAAs have been isolated also flom flied ground beef, pork, and flied meat emulsion (Becher et al., 1988; Felton et al., 1986a; Gry et al., 1986; Skog et al., 1998). Non-polar heterocyclic aromatic amines Mutagenic and/or carcinogenic HAAs have also been classified as polar and non-polar compounds. To obtain a better estimation of the total concentration of HAAs in cooked foods, analyses have been extended to include non-polar HAAs such as 3- amino-1,4-dimethy1-5H-pyrido[4,3-b]indole (Trp-P-l), 3-amino-1-methyl-5H— pyrido[4,3-b]indole (Trp-P-2), 2-amino-6-methyl-pyrido[l,2-a:3’,2’-d]imidazole (Glu- P-l), 2-amino dipyridol [1,2-a:3’,2’-d]imidazole (Glu-P-2), the amino-or-carbolines, 2- amino-9H-pyrido[2,3-b]indo1e (AaC) and 2-amino-3-methyl—9H-pyrido[2,3-b]indole, (MerrC) and the B-carbolines, 1—methy1-9H-pyrido[3,4-b]indole (barman) and 9H- pyrido[3,4-b]indole (norharman). Trp-P-l and Trp-P-2 have been shown to induce liver tumors in mice and rats. The latter has also been implicated in urinary bladder cancer (Takahashi et al., 1993). Chen and Meng (1999) reported that norharman, barman, AaC, and MeAcLC were produced in a model system containing phenylalanine, glucose, and creatinine, when heated at 150 or 200°C. The amino-a—carbolines, AaC and MerrC, were also formed in model systems containing glucose and creatinine at 150 or 200°C. Abdulkarim and Smith (1998) demonstrated, the presence of norharman, harman, and Trp-p-2 on the surfaces of processed meat samples, including flesh pork sausage, bratwurst, Italian sausage, and smoked sausage. They also reported high concentrations of norharman (30.0 ng/g), harman (28.6 ng/g), and Trp-p-2 (1.59 ng/g) 21 in beef steak that was grilled at 240°C for 14 min. Non-polar HAAs have not received as much attention as the polar HAAs because they were thought to be formed exclusively under extreme cooking conditions and were not believed to be found in the Western diet (Lansen et a1; 1990). , Other bet—emaic aromatic amines With the improvements in isolation and identification methodologies, several other HAAs have been identified. TMIP (2-amino-l,5,6-trimethyl- imidazopyridine) and DMIP (2-amino-1,6-dimethy1- imidazopyridine) have been isolated from flied meat products (Becher el al., 1988; Felton el al., 1984). In addition, a number of oxygen-containing HAAs has been identified. A methylimidazo—firropyridine was identified in flied minced beef containing milk and creatine (Knize et al., 1990). Knize et a1. (1991) reported the formation of 2,6-diamino-3,4-dimethyl-7—oxo-pyrano[4,3- g]benzimidazole in a dry-heated mixture of creatine, glutarnic acid and glucose. 4,7,8- TriMeIQx (2-amino-3,4,7,8- tetramethylimidazo[4,5-j]quinoxaline) was identified in a model system containing alanine, threonine, creatinine and glucose (Skog el al., 1992). Several HAAs, identified as 4’-OH-PhIP (2-amino-l-methyl-6-(4-hydroxyphenyl)- i1nidazo[4,5-b] pyridine), 4-CH20H-8-MeIQx (2-arnino-4-hydroxy—methy1-3,8- dimethylimidazo-[4,5-f-quinoxaline) and 7,9-DiMeIQx (2-amino-l,7,9- trimethylimidazo [4,5-g]-quinoxaline), were isolated in broiled, flied beef, or bacteriological grade beef extracts (Kurosaka el al., 1992; Reistad el al., 1997; Wakabayashi el al., 1995). The 4'-OH-PhIP was produced on heating a liquid model system containing tyrosine, creatine and glucose (Wakabayashi el al, 1995). 4- 22 CHzOH-8- MeIQx (Nukaya el al., 1994; Wakabayashi el al., 1995) and 7,9-DiMeIng (Wakabayashi el al., 1995) were identified on heating creatine, threonine and glucose in a model system. The identification of these relatively new HAAs adds to our overall knowledge of HAA formation in foods and illustrates the importance for continuing research in this area. Mutagenicity of Heterocyclic Aromatic Amines Several HAAs have been shown to be extremely mutagenic when subjected to the Ames S. typhimurium mutagenicity test (Felton, 1997; Felton et al., 1986a; Sugimura, 1988). They also exhibit moderate mutagenicity in mammalian cell cultures and cause chromosomal change in mice (F elton and Knize, 1990). Both IQ and MeIQ exhibit mutagenicity in the Ames S. {whimurium mutagenicity test with strains of TA 98, TA 100, TA 1537, TA 1538, and TA 1978 (Kasai et al., 1981a). In contrast to IQ, Mele and 4,8-DiMeIQx, PhIP exhibits relatively weak mutagenic activity in the S. whimurium TA 98, TA 100, and TA 1538. However, PhIP was more mutagenic in cultured mammalian cells (Alink et al., 1988; Felton et al., 1986b). HAAs are metabolically activated by cytochrome P450. Initially, 2- hydroxyarnino-3 -methyl-3H-imidazo[4,5-f]quinoline (N-hydroxy-IQ) is produced as an metabolite from the N-hydroxylation of IQ by cytochrome P450 monooxygenase. This is followed by esterification to an acetyl or sulfate moiety (Okamoto et al., 1981; Paterson and Chipman, 1987; Saito et al., 1985; Snyderwine et al., 1987). Human liver cytochrome P450 isoforms, P4501A2, P450HFLa, and P4503A4, metabolically activate IQ in fetal livers (Kitada et al., 1990, 1991). Aoyama et a1. (1990) and Butler et a1. 23 (1989) reported that cytochrome P4501A2 is also the enzyme for the activation of MeIQ, MeIQx, and 4,8-DiMeIQx. Mutagenicity varies widely among individual HAAs, but can be as high as 661,000 revertants/ug when evaluated using S. whimurium TA98. Aflatoxin B1, a well-documented carcinogen, produces only 6,000 revertants/ug under similar conditions (Table 2). Not all mutagens are carcinogens, however, and carcinogenicity must be determined with animal or cell culture studies. Rodent and primate assays have shown many HAAs to be multisite carcinogens, including IQ, MeIQ, MeIQx, and PhIP, as well as many of the nonpolar HAAs such as Trp-P-l, TIp-P-2, Glu-P-l, G1u-P-2, Ach, and MerrC (Ohgaki et al., 1991). Several studies have shown that 1Q, MeIQ, and MeIQx can produce tumors in the liver, lung, forestomach, small and large intestines, zymbal gland, skin, colon, and the lymph system of mice and rats (Stavric, 1994). Mutation of Ha—ras, Ki-ras, and p53 have been observed in zymbal gland tumors in F344 rats induced by IQ, MeIQ, and MEIQx (Esunri et all, 1989; Ito et al., 1991; Takayama, 1984). PhIP induced mammary gland carcinomas and colon cancer in rodents (Stavric, 1994). A specific mutation of the Apc gene-deletion of a guanine base at 5’-GGGA-3’ site was observed by PhIP- induced colon cancers in rat (Ohgaki et al., 1984, 1986, 1987; Wakabayashi and Sugimura, 1998). Urinary bladder cancers developed by TRP-P-Z in rats and MerrC caused atrophy in the salivary glands and pancreas of rats (W akabayashi and Sugimura, 1998) Formation of Heterocyclic Aromatic Amine in Foods 24 Table 2. Mutagenicity of heterocyclic aromatic amines and typical carcinogens as determined by the Ames Salmonella typhimurium assay (Sugimura and Sato, 1982; Sugimura et al., 1988). Revertants/ug Compound TA98 TA100 IQ 433,000 7,000 MeIQ 661,000 30,000 IQx 75,000 1,500 MeIQx 145,000 14,000 4,8-DiMeIQx 183,000 8,000 7,8-DiMeIQx 163,000 9,900 PhIP 1,800 120 Trp-P-l 39,000 1,700 Trp-P-2 104,200 1,800 Glu-P-l 49,000 3,200 Glu-P-2 1,900 1,200 Orn-P-l 56,800 AaC 300 20 MeaAC 200 120 Aflatoxin Bl 6,000 28,000 AF -2 6,500 42,000 4-N1troquinolin-1-Oxide 970 9,900 Benzo[a]pyrene 320 660 “3“,... N—Nrtrosodiethylamiine 0.02 0.15 N-Nitrosodimethylarnine 0.00 0.23 25 Reactants The mode of formation of HAAs in cooked meats has not been flrlly clarified. Yoshida and Okarnoto (1980) reported that dry heating creatine with either glucose, fatty acids or various amino acids at 100-200°C produced high mutagenic activity, and suggested that these reactants were possible HAA precursors. lagerstad et al. (1983a) suggested that creatine, flee amino acids, and hexose, were precursors of the irnidazoquinolines and imidazoquinoxalines. Chicken and beef contain the same HAAs in similar proportions as does flied ground fish, although in smaller amounts, thus suggesting that HAAs in cooked muscle foods all have similar precursors (F elton and Knize, 1991). Supporting evidence for creatine/creatinine involvement in HAA formation is the low mutagenic activity in cooked food products lacking in creatine (Felton and “Knize, 1990; Reutersward et al., 1987b). Furthermore, lagerstad et al. (1983a) demonstrated a significant increase in mutagenic activity in beef when creatine was spread over the surface before frying. The lack of mutagenic activity in cooked shrimp can also be explained by the low levels of creatinine (Bjeldanes et al., 1982). Free amino acids play an important role in HAA formation. Overvik et a1. (1989) reported that mutagenic activity was detected when amino acids were heated with. creatine at 200 °C. Data summarized in Table 3 indicate that amino acid 1 can produce various HAAs when heated with sugar and creatinine. Sugar involvement in HAA formation was demonstrated by Negishi et al. (1984), 26 a [‘2‘ Table 3. Heterocyclic aromatic amines produced in model systems Compound Yield* Sugar ng Reference Add . Conditions IQ 0.4 pro Dry Yoshida et a1. (1984) 1.0 gly Fru DEG-Water Grivas et a1. (1986) 3.0 phe - Dry Felton and Knize (1990) 13.5 phe glu Dry Felton and Knize (1990) 3.7 ser - Dry Knize et al., (1998) MeIQ nd ala fru DEG-Water Grivas et a1. (1985) IQx 2.7 ser - Dry Knize et a1. (1998) _ 11d thr 81“ Water Skog and Jigerstad (1993) I}: 0.025 phe glu Water Chen and Meng (1999) :r 65 meat meat Water Skog et a1. (2000) ll MeIQx 4.4 gly glu DEG-Water 158m et al. (1983b) ‘3 0.9 ala glu DEG-Water Muramatsu and Matsushima (1985) 1.8 ala rib DEG-Water Muramatsu and Matsushima (1985) 4.2 lys rib DEG-Water Muramatsu and Matsushima (1985) nd thr glu DEG-Water Negishi et a1. (1984) 6-7 gly fru DEG-Water Grivas et a1. (1986) nd ser - Dry Overvik et a1. (1989) nd ala - Dry Overvik et a]. (1989) nd tyr - Dry Overvik et a1. (1989) 4 elv glu DEG-Water Skoe and Jaeerstad (1990) mi phe glu DEG-Water arrow and 151mm (19911 10 ala, thr glu DEG-Water Skog et al. (1992) 8.8-17.9 sly glu Water Johansson and Jagerstad (1993) 7-10 80 8111 Water Skog and Jagerstad (1993) 9 thr 8111 Water Skog and lagerstad (1993) ad sly glu Water Jolmnssonand Iagcrstad (1993) 110 meat meat Water Skog et a1. (2000) 4,8-DiMeIQx nd thr glu DEG-Water Negishi et a1. (1984) 1.9-2.6 ala fru DEG-Water Grivas et a1. (1985) 4.2 ala glu DEG-Water Muramatsu and Matsushima (1985) 1.5 ala rib DEG-Water Muramatsu and Matsushima (1985) 26.1 lys rib DEG-Water Muramatsu and Matsushima (1985) nd sly glu DEG-Water Skog and Iagerstad (1990) nd phe glu DEG-Water Skog and Jaeerstad (1991) 36 ala, thr glu DEG-Water Skog et a1. (1992) 30 thr glu Water Skog and Jaeemtad (19931 nd gly glu Water Iohansson et a1. (1993) 7,8-DiMeIQx 1.1 gly glu DEG-Water Negishi et a1. (1984) Nd 81y glu DEG-Water Skog and Jagerstad (1990) 27 Nd gly G1“ ' Water Johanssonandlfiaerstad (19931 4,7,8- 6 ala, thr Glu DEG-Water Skog et al. (1992) TriMeIQx PhIP 3.6 phe glu DEG-Water Shioya et a1. (1987) 735 phe - Dry Felton and Knize (1990) 560 phe glu Dry Felton and Knize (1990) Nd phe - Dry Overvik et a1. (1989) Nd leu - Dry Overvik et a1. (1989) 20-9 phe 811’“ DEG-Water Skog and Jigerstad (1991) 6-4 phe - DEG-Water Skog and Jagcmtad (1991) 0.06 phe glu DEG-Water Manabe et a1. (1992) 14 meat meat Water Skog et a1. (2000) Norharman 0.035 phe glu Water Chen and Meng (1999) Harman 0.035 phe glu Water Chen and Meng (1999) As adapted from Skog, 1993. 'Yield is in mnol/Irmrol creatin(in)e. Dry = dry heating at 180 or 200°C for 1 hour. DEG-Water = reflux boiling in diethylene glycol/water (5: 1) or 14% water. Water = heated in water in closed metal tubes at 180°C for up to 30 minutes. Amino acids: pro = proline, gly = glycine, phe = phenylalanine, ser = serine, ala = alanine, thr .= threonine, lys = lysine, tyr = tyrosine, 1eu = leucine. Sugars: fl'u = fluctose, glu = glucose, rib = ribose. nd'= not determined but its role as a precursor, still remains unclear. Many studies have addressed the impact of various sugars on HAA formation in model systems containing creatine/creatinine, and amino acids (Manabe et al., 1992; Muramatsu and Matsushima, 1985; Skog and lagerstad, 1990, 1991). The incorporation of carbon atoms from 14C—labelled glucose into IQx, MeIQx and 4,8-DiMeIQx under model system conditions confirmed that glucose is an important precursor for HAA formation (Skog and lagerstad, 1993). Effect of cooking time and temperature on HAA formation A number of investigators have demonstrated that HAA formation increases with increasing temperature of cooking (Balogh et al., 2000; Bjeldanes et al., 1983; Chen, 1988; Hatch et al., 1982; Knize et al., 1994; Sinha et al., 1998; Spingam and 28 Weisburger, 1979). Cooking methods that employ higher heating temperatures generally induce greater HAA formation than do lower temperature methods (Sinha et al., 1998). Several investigators also have observed that there is a rapid increase in HAA formation with increasing cooking time (Balogh et al., 2000; Chen, 1988; Knize et al., 1994; Sinha et al., 1998). However, there is a lag period of 0 to 2 min during the flying of ground beef patties when no HAA formation is observed. This is the time required for the meat patty crust surface to reach temperature of 100°C and above (Knize et al., 1994). Recent work by Balogh et al. (2000) confirmed the increase in HAA formation in ground beef patties with increasing time and/or temperature of flying. There was a pronounced increase in the formation of HAAs when the temperature was raised from 175°C to 200°C and 225°C. Concentrations of total HAAs formed at 225°C were six times greater than corresponding quantities formed at 175°C. Concentrations of total HAAs also increased 2-5 times when cooking times were increased flom 6 min to 10 min. The most abundant HAA in flied ground beef is PhIP. Balogh et al. (2000) demonstrated that concentrations of PhIP in the beef patties flied at 225°C for 10 min were approximately 10 times greater than those of MeIQ. Arvidsson et al. (1999) reported that a longer time and a higher cooking temperature are necessary to produce PhIP in model systems containing creatinine, glucose, and amino acids because IQx, MeIQx, and DiMeIQx have lower activation energies of formation than PhIP. Chgmg' try of HAA formation 29 'P’W'E‘ 2.1-. 1.1:, A possible route of HAA formation is through intermediates of the Maillard or non-enzymatic browning reaction (P0wrie et al., 1981; Shibamoto et al., 1981; Spingarn and Garvie, 1979; Wei et al., 1981). The Maillard reaction takes place in food through a series of reactions between reducing sugars such as glucose and fluctose, and compounds possessing available amino groups such as amino acids, peptides, and proteins. The combining of an available amino group and a reducing sugar produces a glycosylamine which undergoes an Amadori rearrangement to produce a l-amino-2- keto sugar (Figure 2). This intermediate may then be broken down into 2- and 3- carbon fragments by two pathways (3 -deoxyhexosone and methyl or-dicarbonyl routes), leading to the formation of a variety of compounds such as aldehydes, ketones and melanoidin pigments. Pyrazine and pyridines can be produced flom the interaction of the a—dicarbonyls flom the Maillard reaction with amino acids, the so-called Strecker degradation (Rizzi, 1994). Aldehydes, flrranones, sulfur-containing heterocyclic compounds, pyridines, pyrazines, and pyrroles are volatile compounds produced via the Maillard reaction which contribute to the flavor and aroma of cooked foods. Melanoidines are color compounds formed during the Maillard reaction which contribute to brown color formation in breads and cooked meats (Rizzi, 1994). A mechanism for the early stages of the Maillard reaction identifying the Amadori rearrangement as a key step was proposed by Hodge (1953). This was subsequently questioned by Narniki and Hayashi (1981). They reported the formation of the N,N’-disubstituted pyrazine cation by early carbon fragmentation prior to the Amadori product. They demonstrated that radical products are formed by the 30 RNH HOH HC:O +RNH, "H20 . 2:?— ( Homo 'T—-—-— " ' ". ( OH)n (CHOH)n 1m ' 10“ OH cmoa ' aldoae Schifi‘ base / + RNH RNH RNH W +( OHM" ‘__'(IIOHIHOI§__ n H H—J H (H0100 on . H203 on N-suhstttated N-substitutad \eation of / glycosylamine Mom-mine Schlfl’bue mm mm | . + OH :———> C: O N-substrtuted l-amino-l -deoxy (3 Eli-1 (a 10mm z-ketose, keto form OR Amadori product) enol form Figure 2. Initial steps of the Maillard reaction (Hodge, 1953). 31 condensation of two molecules of the two-carbon enaminol compounds which might be formed either directly from Schifl’ 3 base products or indirectly through the reaction of glycoaldehyde with amino compounds (Figure 3). They also reported that C2 and C3 fragments were produced prior to the Amadori rearrangement by a reverse-aldol reaction of the glycosylamine leading to the formation of a glycolaldehyde alkylimine. The pyridine radical would result flom the interaction of glyoxal monoalkylirnine with glyoxal. This compound can then be oxidized to form a glyoxal monoalkylirnine, which produced less flee radical and reacted more slowly than the glycoaldehyde system (Namiki and Hayashi, 1981, 1983). Glycoaldehyde is very efl‘ective in facilitating rapid and extensive radical formation compared to glyoxal. A possible mechanism of HAA formation through the Maillard reaction pathway was proposed by lagerstad et al. (1983b). They assumed that pyridines and pyrazines, formed via the Maillard reaction, react with an aldehyde to form the quinoline or quinoxaline moiety which is central to the structure of HAAs. Creatine is dehydrated by heat and cyclized to creatinine which reacts with the aldehyde to form an IQ-or IQx- type HAA (Figure 4). This theory was confirmed in a model system study in which creatinine, .glycine or alanine, and glucose, dissolved in diethylene glycol containing 14% water, were boiled under reflux at 130 °C for 2 hr. The mixture showed high mutagenic activity, whereas heating the. reactants in ' combinations of two produced only weak, if any, mutagenic activity. The addition of sYnthetic pyridines or pyrazines to the reaction mixture increased the mutagenic activity by 50% (Jagerstad et al., 1983b). Grivas et al. (1985) utilized a similar model system, employing fructose as the sugar and glycine as the amino acid. They isolated MeIQx 32 H—C=N—R 1m ..'_... JH— OH C._.._.N—R H— C=N—+R H—C-—N——R IF—‘C—OH H—C_OH H—‘C—OH F ——-——-> n——> T -—-—_ H—C—'— H H—CZO H C O“ l H—c—N—R R. R1 1,1- RI HC—OH + R N _——-—‘. T——_ N. R Browning (Melanoidin) Figure 3. A suggested pathway of browning in the Maillard reaction through a flee radical (Namiki and Hayashi, 1981). 33 and IQ, thus ofl‘erred further confirmation of the precursors needed to form HAAs. Support for the condensation of aldehydes with creatinine was proposed by Jones and Weisburger (198 8) who reported the formation of different IQ-like mutagenic (products I through reactions between creatinine and different aldehydes. This model involved the condensation of two molecules of acetaldehyde with creatinine in one step to form IQx- type compounds. They found no mutagenic activity in model systems containing 2- vinylpyrazine and creatinine, an observation that may indicate that aldehydes are necessary for HAA formation. In a later study, Nyhammer (1986) proposed that HAAs were formed by an aldol-type condensation between an aldehyde and a pyridine or pyrazine molecule, followed by the cyclic addition of creatinine to yield either an imidazoquinoline or an imidazoquinoxaline. Namiki and Hayashi (1975, 1980) proposed that HAA formation may involve a free radical process because flee radicals have been shown to occur in the Maillard reaction, both prior to and following the Amadori rearrangement. Milic et al. (1993) provided filrther support with electron spin resonance studies of HAA formation which confirmed pyrazine/pyridine flee radical cation involvement. The pyrazine and pyrazine flee radicals were suggested to be precursors of HAAs. Reduction of Heterocycfic Aromatic Amine Formation in Foods Many studies have shown that compounds such as sugars and other carbohydrates, soy protein concentrates and defatted glandless cottonseed flour, antioxidants, tea phenolics, and several compounds flom fluits and vegetables inhibit 34 “'1 ‘0 .- a ‘ NH2 NHs+ HN R—CH C6H1206 HOOC N - \COO' \/ \CHs Heme IAmino Acid I I Creatine*I 41,0 NHZ Y z N=( ’ O + OHG—R + A” 0 \CH x N f CH3 Aldehyde 3 Pyridine , Creatinine or Pyrazine NHZ N/ N_CH3 Z “(O O X N I IQ or IQ: Compound Figure 4. Theoretical reaction pathway for formation of IQ and IQx compounds. R, X, and Y may be H or CH3: Z may be CH or N (Ja'gerstad et al., 1983b). 35 HAA formation in food and model systems '(Botting et al., 1999). WWI—M Sugars will impact HAA formation in food and model systems. Taylor et al. (1985) reported that glucose, when added to a beefstock supernatant at a concentration four times greater than that of creatine, reduced the overall mutagenicity of the heated system. Similarly, Skog and Jagerstad (1991) demonstrated that high amounts of sugar compared to the other two reactants (creatinine and phenylalanine) in model Systems containing glucose, creatinine, and phenylalanine inhibited HAA formation. When sugars were present in equirnolar or greater concentrations than creatin(in)e, HAA formation was notably reduced. Greatest mutagenicity was produced when the glucose level was one half that of creatinine or the amino acid. These results, observed only when amino acids were present in the reaction mixture, indicate a reaction between Maillard reaction products such as S-hydroxymethyl-Z-flrrflrral (HMF) and creatinine. Thus, less creatinine would be available to form HAAs (Skog and lagerstad, 1990). The inhibitory effect of sugars, when added in excess, has also been studied in meat systems. Skog et al. (1992) demonstrated that glucose and lactose flom milk powder, when added at concentrations up to 4 %, reduced mutagenicity by 34 to 76%. The greatest inhibitory effect was achieved by golden breadcrumbs when they were added in combination with glucose or lactose in meat systems. $11 protein concentrate and defatted glandlgs cottonm flour 36 Soy protein concentrates have been reported to reduce mutagenicity in flied beef patties by 24% (Wang et al., 1982). This reduction was attributed mainly to volumetric effects including the reduction of interactions arnong the beef components, and the reduction of the amount of beef that came into contact with the flying surface. Some consideration was given also to the possible inhibitory role of chlorogenic acid, a naturally occuring polyphenolic antioxidant in soy protein concentrates (Pratt and Birac, 1979; Rappaport et al., 1979; Smith and Circle, 1978). Another possible factor was the binding of soy protein concentrate with water, thus limiting the movement of the reactants to the surface of meat in contact with the flying system. .Defatted glandless cottonseed flour, when added at a level of 5% (w/w) to ground beef before flying, reduced the overall mutagenicity of the cooked meat (Rhee et al., 1987). As quercetin derivatives are the major flavonoids present in defatted glandless cottonseed, some consideration was given to their possible role as inhibitors of HAA formation. However, it was not clarified whether the reduction of mutagenicity was due to volumetric efl‘ects or to the intervention of specific cottonseed components. Phenolic antioxidants It has been shown that butylated hydroxyanisole (BHA) will reduce the nrutagenicity of flied ground beef patties. Chen et al. (1992) confirmed the results of Wang et al. (1982) and further reported that BHA, propyl gallate and tertiary butylhydroquinone reduced the overall concentrations of IQ-like compounds in flied ground beef by approximately 80-90%. More recently, Faulkner (1994) reported that BHA (0.1% based on the fat content) reduced the mutagenicity in cooked beef patties 37 ..1 flom 7000 revertants/100g raw meat to 2800 revertants/lOOg (raw meat basis) as determined by the Ames S. typhimurium assay. ‘ Vitamin E has been extensively studied as an inhibitor of HAA formation in meat system. Chen et al. (1992) demonstrated that vitamin E (1% based on the fat content) reduced the concentration of IQ-like compounds by 50%. Balogh et al. (2000) reported that vitamin E (1% and 10% based on the fat content) reduced PhIP concentrations in flied beef patties by 69% and 72%, respectively. The direct addition of vitamin E (1% based on fat content) to the surface of the beef patties reduced MeIQx and MeIQ formation by 45% and 76%, respectively. These results clearly establish vitamin E as an effective inhibitor of HAA formation in ground beef patties. Tea phenolics Tea polyphenolic compounds such as epigallocatechin gallate (EGCG), epicatechin gallate, and epigallocatechin, have been established as potent antioxidants (Chen et al., 1990; Ho et al., 1992; Sichel et al., 1991; Sorata et al., 1984; Terao et al., 1984). The efl‘ects of black tea, green tea and the polyphenolics, theaflavine gallate (TFG) and EGCG, on the formation of HAAs were studied by Weisburger et al. (1994). They demonstrated reduction of HAA formation in model systems containing creatinine, glycine and glucose. Black tea had a substantial effect in lowering the formation of both MeIQx and PhIP (flom 9350 to 7340 revertants/plate and from 6530 to 2070 revertants/plate, respectively). The effect of green tea in reducing the formation of mutagenic compounds was efl‘ective only for PhIP (flom 6530 to 2180 revertants/plate). They also demonstrated that EGCG and TFG reduced mutagenic 38 activity flom 74% to 83% for both MeIQx and PhIP. Oguri et al. (1998) investigated the inhibitory effect of green tea catechins and EGCG on formation of HAAs and their antimutagenicity activity. Green tea catechins and EGCG reduced MeIQx formation by 21% and 35%, respectively. The antimutagenic effects of green tea catechins and EGCG on MeIQx toward S. whimurium TA98 were 29% and 46%, respectively. Many studies have shown the relationship between antioxidant activity and antimutagenicity of green tea, pouching tea, oolong tea and black tea (Yen and Chen, 1995). The antimutagenic effect of tea extracts on IQ toward S. whimurium TA98 and TA100 was correlated with their reducing power and scavenging effect on the hydroxyl radical. The antioxidant effect of tea extracts was well correlated to their antimutagenicity in some cases, but varied with the mutagenic and antioxidative properties. Dashwood et al. (1999) showed that both green tea (2% w/v) and black tea (2% w/v) reduced mutagenicity against the IQ compound. N-hydroxy-IQ, a direct- acting mutagen in the Ames assay, was inhibited by individual components of tea such as epigallocatechin-B-gallate (EGCG) and epigallocatechin (EGC). Further, they also demonstrated the modulation effect on HAA metabolism. NADPH-cytochrome P450 reductase and MO-acetyltransferase, enzymes for metabolic activation of HAAs, were inhibited by tea. Studies in vivo established that tea also induced cytochrome P450 and Phase II enzymes with the rapid metabolism and excretion of HAAs. History and Therapeutic Effects of Garlic Garlic has been cultivated since antiquity and has been used as foodstuffs and medicines. Botanists call it Allium sativum L., and the origin of the genus name 39 remains unknown. It is possibly derived flom Latin word olere, “to sme ” because of its strong odor or it is derived flom the Greek word hallestai, “to leap out” (Milner, 1996). Garlic has approximately 600 known species distributed over Europe, North America, North Aflica, and Asia, and the cultivated form of garlic, Allium sativum L., presumably originated flom central Asia (Lawson, 1996). Large amounts of garlic are produced in Egypt, India, China, and South Korea. Recently, China has become a major user and producer of garlic. Argentina is the notable main producer in South America. The “Garlic Capital” of the US. is Gilroy, CA because it produces around 80 to 90% of the garlic consumed in the US. Garlic has captured a secure popular position with consumers due to its various alleged therapeutic effects and common use. It was formerly popular, and still is, to some extent as a carminative for dyspeptic problems and diarrhea, an antimicrobial for bacteria, a firngal, and viral infections, as well as a vermifuge for intestinal parasites (Reuter and Sendl, 1994). In recent years, garlic has become highly valued because of its excellent efl‘ectiveness toward arteriosclerosis, its ability to lower serum cholesterol and triglyceride levels, and its hypotensive, anticarcinogenic, and antidiabetic effects (Klitchevsky, 1991). Garlic may also inhibit thrombocyte aggregation and activate fibrinolysis (Kendler, 1987; Orth-Wagner, 1986; Weiss, 1986). The US. Department of Agriculture reported that U. S. garlic consumption in 1989 was 1.0 pounds per capita, and it has soared to 3.1 pounds in 1999 (Lucier and Lin, 2000). 40 The composition of garlic The composition and chemistry of garlic has been extensively reviewed because of its popularity and various health benefits. Garlic has been sold in various fornrs such as bulbs, picked cloves, crushed or chopped cloves, spice powders, and garlic salts. Also, garlic supplements are sold as powder tablets or capsules with various coatings. Due to chemical and enzymatic changes that take place during processing or preparation steps, different forms of garlic contain different compounds (Lawson, 1996). The general. composition of garlic is shown in Table 4. The water content of garlic is about 65%, which is relatively lower than most fluits and vegetables (80-90%). The bulk of the dry weight is composed of carbohydrates, sulfur compounds, protein, and flee amino acids (Lawson, .1996). Sulfur compounds in gyflc Many garlic studies have focused on the sulfur compounds because of their high concentrations relative to those in fluits and other vegetables (Figure 5). Various reports have cited the health promoting effects of sulfirr compounds including antibacterial (Deshpande et al., 1993; Hughes and Lawson, 1991; Shashikanth et al., 1986), antifungal (Ghannonoum, 1988; Hughes and Lawson, 1991; Yoshida et al., . 1987), antiarteriosclerotic (Mohammad and Woodward, 1986), antithrombotic (Lawson et al., 1992), and blood lipid-lowering activities (Kamanna and Chandrasekhara, 1984; Plengvidhya et al., 1988; Sitprija et al., 1987). A German scientist, Wertheim (1844), discovered that steam distillation of cnlshed garlic produced strong smelling oil which consisted exclusively of organosulfur 41 Table 4. The general composition of garlic. Component Amount (%fresh weight) Water ' 62-68 Carbohydrates 26-30 Protein 1.5-2.1 Amino acids: common 1-1 .5 Amino acids: cysteine sulfoxides 0.6-1.9 y-Glutamylcysteine 0 . 5-1 .6 Lipids 0.1-0.2 Fiber 1.5 Total sulfur compounds 1.1-3.5 Sulfur 0.23-0.37 Nitrogen 0.6-1.3 Minerals 0.7 Vitamins 0.015 Saponins 0.04-0.11 Total oil-soluble compounds 0.15 (whole)-0.7 (cut) Total water-soluble compounds 97 Table compiled using information flom Lawson et al. (1991), Lawson (1993), Pentz et al. (1990), and Ueda et al. (1991). 42 5' Pas riiiiiriiii 0 1 2 3 mg/g Fresh weight Figure 5. Sulfur content of common fruits and vegetables (Nielsen et al., 1991). 43 . . .. . _ . . . . ,_ _ . . .. . . ‘ .1 , ,_ t .- I . .. . I" an .11 1...,” . . .,.,,2,‘ ,. , I _ , _¥1w$1*~fiW""“-W}fi1: :r, ., .r . rm 1 _ ,, .Iut. M. , ”Us”, . ‘ ‘ 1 . compounds. He also determined these compounds had a basic formula of Camps and named the hydrocarbon group “allyl” after Allium sativum. However, almost 50 years later, Semmler (1892) flactionally distilled the oil and identified specific compounds. He correctly determined the formula of allyl to be C3H5 instead of CsHm and found the oil contained 60% diallyl disulfide (DAD), 20% diallyl trisulfide, 10% diallyl tetrasulflde, and 6% allyl propyl disulfide. The allyl propyl disulfide content was later shown to be an error since S-propyl compounds are not produced by garlic (Block et al., 1992, 1993; Lawson et al., 1991). Almost 50 years afier Semmler’s work, Cavallito and colleagues (Cavallito and Bailey, 1944; Cavallito et al., 1944, 1945) reported that the antibacterial activity of crushed garlic clove was due to the presence of an oxygenated sulfur compound which possessed the odor of fleshly cut garlic. This compound was named allicin. Cavallito et al. (1945) concluded that it must be formed on crushing garlic, and it was the precursors of the diallyl sulfides present in the oil of steam-distilled garlic. Stoll and Seebeck (1947) reported that alliin, the parent compound of allicin, had no antibiotic activity unless converted to allicin by an enzyme, alliinase. The thiosulfinates present in crushed garlic are transformed at room temperature to diallyl trisulfide, diallyl disulfide, and allyl methyl trisulfide (Iberl et al., 1990). Work in Finland on onions (Virtanen, 1965, 1969; virtanen et al., 1992) and ”s—labelcd garlic studies (Sugii et al., 1964; Suzuki et al., 1961, 1962), indicated that garlic contains nine different 7- glutarnylpeptides, six of which contain the sulfur containing amino acid, cysteine. The .most abundant y—glutamylcysteine in garlic, y—glutamyl-S-trans-propenylcysteine, was not discovered until almost 30 years later (Lawson and Hughes, 1990; Lawson et al., 44 1991). The sulfur content of garlic is approximately 1.0% of its dry weight (0.35% of its flesh weight) (Pentz et al., 1990; Ueda et al., 1991). Alliin, allicin, and the two main y—glutamylcysteines, constitute the majority (about 72%) of the sulfirr compounds in whole or crushed garlic. Sixteen nonprotein organosulfur compounds have been found in whole cloves and 23 in crushed cloves (Table 5). Three minor compounds (methionine, 'y-glutamylmethionine, and thiamine) out of the 16 nonprotein organosulfllr compounds present in whole cloves do not contain the amino acid cysteine. Figure 6 shows the formation of allicin by the action of alliinase and the transformation of the principal thiosulfinates of crushed garlic. The minor organosulfur compounds consist mainly of methyl and l-propenyl homologs of alliin and allicin, and y—glutamyl-S-methylcysteine (which comprises about 13% of the total sulfur). Trace amounts (less than 0.1 mg/g) of a few other related compounds (8 in whole cloves, 11 in crushed cloves) are also found. Protein-soluble and insoluble-sulfllr, and inorganic sulfate, comprise about 9% and 5% of the total sulflrr (Lawson, 1993). The sulfiir compounds of garlic constitute about 86% of the total sulfur of garlic cloves and about 98i2% of the total nonprotein organosulfllr compounds. Role of Sulfur Compounds in the Biotransformation of Xenobiotics ' and the Inhibition of Heterocycfic Aromatic Amine Formation Sulfur compounds have been reported to play a key role in the biotransformation of xenobiotics by actively participating in their detoxification and also by inhibiting the 45 Table 5. Sulfllr compounds in whole and crushed garlic cloves (Lawson, 1996). Compound Whole Garlic Crushed Garlic (mg/g flesh weight) gating enIyl-Iflsteine Sulfgxiges S-Allylcysteine sulfoxide (alliin) 54 4 11 db S-Methylcysteine sulfoxide (methiin) 0 5_2 0 nd S-trans-l-Propenylcysteine sulfoxide (isoalliin) 0'2_1'2 n d S-Propylcysteine sulfoxide ' rid nd Cy°l°amm 0.5-1.5 0.5-1.5 1-L-Glutamxl-S-a_ll_g enIXI-L-cysteines x-Glutamyl-S-trans-l-propenylcysteine 3-9 3-9 x-Glutamyl-S-cis-1-propenylcysteine 0.06-0. 15 0.06—0. 15 1-Glutamy1—S-allylcysteine 2-6 2-6 x-Glutamyl-S-methylcysteine 0. l-0.4 0. 1-0.4 x-Glutamyl-S-propylcysteine nd nd Thiosulfinates nd 2-6 Allyl 2-propenethiosulfinate (allicin) nd 03-1.5 Ally methyl thiomlfinates nd 0.05-1.0 Ally trans-l-propenylthiosulfinates nd 0.02.0.2 Methyl trans-l-propenylthiosulfinate nd 0_05.o_1 Methyl methanethiosulfinate Others nd nd , nd nd Cysteine nd nd We nd nd Glutathione, reduced nd nd Glutathione, oxidized nd nd 1-Glutamyl-S—allycysteine sulfoxide nd nd r-Glutamyl-S-Uans-l-pmpenylcysteine sulfoxide 0.02-0. 12 002-0. 12 z-Glutamyl-S-methylcysteine sulfoxide nd nd 1-Ghrtamy-methionine tr tr I-Glutamylsysteine, reduced 0.09 0.09 S-2-carboxypropylglutathione 11:: mm cot; lutamyl-S- cine - ISilidethycysteine nd-0.026 nd-0.026 S-l-Propenyl cysteine nd 0.002 S-Allylcysteine 0.02 0.02 S-Allylmercaptocysteine 0-002 “(I‘D-001 Methionine nd nd-0.001 Thiamine 0.03 (yield) <0.03 Allithiarnine nd, 0.01 nd, 0.01 Scordinins 0.3 0.3 Sulfolipids 0.6 0.6 Protein, soluble 0-5 0-5 OI- wr-t‘mu - m {I t! A I. I i Nl'b o 4¢¢\\//8 COOH Alliin (Crushing 0f Alliinase mature bulbs) (I) Ws\s/CH.3 Allicjn Allyl methanethiosulf'mate s s s S \ 101 W ‘SM W ‘s’ W L? ARA/RSV Was/Y.” Diallyldisulfide Dallyltrisulndc 2virryr4m3dithiin EAjome SAW 999* S S st E _ S WSH Ws‘s’ma W ‘s’ ‘01": W W ms’ \A Allylmefllyl dadfide Allylnxefllyl trisulfide 3-mel-4H-12-dithiin ZAjoene Allyl are-calm Figure 6. Fonmnor ofallicin bytheaction ofalliirraseand transformation ofthe prina'pal thiosulfinates ofaushed garlic (Blocket al., 2001). 47 action of mutagens, carcinogens, and other toxic compounds (Friedman and Molnar- Perl, 1990). Many studies have shown the effect of sulfur compounds on the biotransformation of xenobiotics by detoxification and modulation of mutagenesis, carcinogenesis, and other toxic compound metabolism (Sheen et al., 1999). DAD inhibited benzo[a]pyreneinduced forestomach tumors, pulmonary adenoma and skin carcinogenesis (Singh and Shukla, 1998; Spamins et al.,‘1988). Surh et al. (1995) demonstrated that DAD reduced vinyl carbamate and N—nitrosodimethylamine (NDMA)-induced mutagenesis which correlated with inhibition of P450 2El-mediated ‘p-nitrophenol hydroxylation and NDMA N—demethylation. Friedman et al. (1982) reported that cysteine and other related thiols inactivated the mutagenecity of aflatoxin in in vitro experiments. De Flora (1989) showed that the N—acetyl-cysteine dramatically decreased urethane-induced tumor formation in mice. Troll (1986) reported that a sulfur-rich protein called the Bowman-Birk inhibitor suppressed N-nitrosamine—induced carcinogenicity in the digestive tract of rats. They suggested that the possible eflecfiveness of garlic sulfur compounds in preventing the formation of toxic compounds is by trapping intermediates, and by preventing activation of biologically active forms. There have been several reports of HAA inhibition in meats by onion and garlic extracts. .Kato et al. (1998) reported reduction of mutagen formation (74%) in hamburger by the addition of onion juice. Murkovic et al. (1998) found that garlic reduced IQ, MeIQ, MeIQx, Di MeIQx, and PhIP formation in flied ground beef by 32%, 71%, 40%, 78%, and 54%, respectively. They suggested that the effect of garlic may be due to sulfllr compounds in the essential oil of garlic. Salmon et al. (1997) 48 demonstrated that marinating chicken breasts for 20 min before grilling reduced PhIP formation by 92-99%. While the marinade contained olive oil, brown sugar, cider vinegar, lemon juice, garlic, salt, and mustard, the inhibitory effect was not assigned to any particular ingredient. Tsai et a1. (1996) demonstrated that addition of sulfur compounds (DAD and dipropyl disulfide (DPD)) to a pork meat juice model system before heating reduced the formation of Maillard reaction products which correlated with a decrease in mutagenicity. They reported that sulfur compounds showed various dose-dependent responses in reducing the mutagenicity of extracts of boiled pork juice by inhibiting IQ mutagen formation. Recently, Trompeta and O’Brien (1998) reported that various sulfur compounds including glutathione, L-cysteine, L-cystine, and deoxyalliin inhibited the formation of mutagenic compounds in a model system containing glucose, glycine, and creatinine which was heated in a diethylene glycol reflux system. They suggested several possible mechanisms of inhibition of HAA formation by sulfur compounds. These compounds could act as reducing agents, scavengers of flee radicals, strong nucleophiles which can trap electrophilic compounds and intermediates, precursors for intracellular reduced glutathion, and inducers of cellular detoxification. These investigators further proposed that addition of sulfur compounds could possibly lead to decreasing amounts of HAA formation through the competitive reaction between sulfur compounds and amino acids for glucose. This proposition is supported by the results of Arvidsson et al. (1999), which showed the rapid depletion of glucose in the early stages of the reaction. Sulfilr compounds have been shown to react with glucose to produce several of volatile compounds (Yu et al., 1994). Thiazoles, especially 2—acetylthiazole, were 49 I1- predominant volatile interaction products of alliin and glucose, whereas pyrazines, especially 2,5-dimethyl-, methyl-, and trimethylpyrazine, were found to be the predominant volatile interaction products of deoxyalliin and glucose. 50 CHAPTER TWO Inhibition of heterocyclic aromatic amine formation in fried ground beef patties by garlic and its sulfur compounds ABSTRACT The effects of garlic and selected organosulfirr compounds (diallyl disulfide, dipropyl disulflde, diallyl sulfide, allyl methyl sulfide, allyl mercaptan, cysteine, and cystine) on the formation of heterocyclic aromatic amines (HAAs) in fried ground beef patties were evaluated. Minced garlic cloves (approximately 4.8-16.7% w/w) or organosulfur compounds (0.67 mmol) were added directly to ground beef. Patties (100g) were flied at 225°C (surface temperature) for 10 nrin per side. Two patties were flied for each replication, and five replicates were analyzed for each treatment. For each replicate, four sub-samples were analyzed (two unspiked for concentration and two spiked for recovery of HAA standards). The volatile sulfur compounds significantly (p < 0.05) reduced the concentrations of 2-amino-1-methyl-6-phenylimidazo[4,5-b] pyridine (PhIP) (46-81% reduction), while average reductions of 35, 22, and 71%, were achived by cystine, cysteine, and whole garlic, respectively. Concentrations of 2- amino-3,8—dimethylimidazo-[4,5-j]quinoxaline (MeIQx) were reduced 34 to 67% by the volatile sulfilr compounds, and 25, 19, and 63% (p<0.05) by cystine, cysteine, and whole garlic, respectively. These studies confirm that garlic and some organosulfllr compounds have the potential to reduce HAA formation in cooked beef patties. 51 __ [—— INTRODUCTION The human diet is a complex mixture of organic and inorganic substances that not only provide nutritional benefits, but also play a role in the causation and modulation of human health risk (Thomson, 1999). Many mutagens and carcinogens have been isolated and identified in foods. Recently, several foods and constituents of foods have been investigated for their inhibitory or promotional efl‘ects on carcinogenesis (Knize et al., 1999; Weisburger, 1991). I Heterocych aromatic amines (HAAs) are formed in muscle foods when cooked at temperatures generally in excess of 150°C. Several have been identified in cooked fish and meat and are believed to result flom the interaction of creatine/creatinine, sugars and flee anrino acids. These compounds, also called aminoirnidazoazaarenes, can be broken down into four categories: quinolines, quinoxalines, pyridines, and furopyridines. The most commonly found HAAs in foods are IQ (2-amino-3- methylimidazo[4,5-f]-quinoline); MeIQ (2-amino-3,4dimethylimidazo[4,5-f]- quinoline); MeIQx (2-amino-3,8 dimethylimidazo [4,5-fl—quinoxaline); 4,8 DiMeIQx (2-amino-3,4,8 trimethylimidazo[4,5-j]-quinoxaline); and PhIP (2-amino-1-methyl-6- phenylinridazo [4,5-b]-pyridine) (Knize et al., 1999; Skog, 1993; Wakabayashi and Sugimura, 1998). Cooking time and temperature, and method of cooking, all influence the formation of HAAs in cooked meat products (Knize et al., 1994, 1999). Generally, those methods where meat is in direct contact with the heating surface or flame, such as in pan-frying and grilling, produce the greatest quantities of HAAs. The formation of HAAs in cooked meat can be reduced by the addition of selected compounds or 52 ingredients such as'vitamin E (Balogh et al., 2000), cherry tissue (Britt et al., 1998), tea polyphenolic compounds (Yen and Chen, 1995), soy protein concentrate (Wang et al., 1982) and defatted glandless cottonseed flour (Rhee et al., 1987). Garlic has been cultivated since antiquity and has been used as a foodstufl‘ and for medical purposes. A variety of garlic-based health products are now readily available on the market. In recent years, garlic has become highly valued because of its impact on arteriosclerosis (Klitchevsky, 1991), its ability to lower serum cholesterol (Reuter and Sendl, 1994) and triglyceride levels (Weiss, 1986), and for its hypotensive (Kendler, 1987), anticarcinogenic (W attenberg et al., 1989) and antidiabetic properties (Augusti and Sheela, 1996). Garlic also inhibits thrombocyte aggregation (Nishimura et al., 1988) and activates flbrinolysis (Weiss, 1986). Many food preparation methods in the home include the cooking of meats with various vegetables and spices that contains organosulfur compounds. However, there is not much information on the effect of these compounds on HAA formation during the cooking of meats. The objective of this study was to evaluate the effectiveness of garlic and individual organosulfur compounds present in garlic as inhibitors of HAA formation in cooked ground beef patties. 53 MATERIALS AND METHODS Safety Heterocyclic aromatic amines are mutagenic/carcinogenic and should be handled with appropriate safety precautions including the use of goggles, latex gloves and efficient fume hoods. Materials Diallyl disulflde (DAD), dipropyl disulfide (DPD), diallyl sulfide (DAS), allyl methyl sulfide (AMS), allyl mercaptan (AM), cysteine, and cystine were purchased from Fluka Chemical Co. (Buchs, Switzerland). The HAA standards (MeIQx, 4, 8- DiMeIQx, and PhIP) were obtained flom Toronto Research Chemicals (Toronto, Canada). The HAA standard (FEMA-Flavor and Extracts Manufacturer’s Association) and the internal standard, caffeine, were gifis flom Dr. Mark Knize, Lawrence Livermore National Laboratory, Livermore, CA. The FEMA standard contained IQ, MeIQ, MeIQx, DiMeIQs, and PhIP, each at 5 ng/uL. Propyl-sulfonic acid (PRS) Bond- Elut columns (500 mg) and C18 cartridges (100 mg) were purchased flom Varian Inc. (Harbor City, CA). Extrelut-20 columns and Extrelut diatomaceous earth were obtained flom E.M. Separations Technology (Gibbstown, NJ). All other chemicals were of analytical grade and were purchased flom Fisher Scientific (Fair Lawn, NJ). Freshly ground beef (approximately 15% fat) was obtained flom a local supermarket and used within one hour of purchase, or stored at -20°C until required for 54 flying. Fat content was determined by the method of Folch et al. (1957). Fresh garlic was also purchased from a local supermarket. Preparation of ground beef patties Patties were prepared from ground beef as follows: control patties (ground beef patties containing 1 mL of methanol) and patties containing various quantities of minced garlic cloves and organosulfur compounds added as separate treatments two hours prior to flying. Minced garlic cloves (4.5 - 18.2%, w/w) or organosulfur compounds (0.17 - 1.01 mmol DAD, DPD, DAS, AMS, AM, cysteine, and cystine) were mixed/dissolved in 1 mL methanol and added directly to the ground beef, and then mixed in a Keebler mixer (Keebler Inc., Chicage, IL) for 2 min. Each patty (100g) was formed by placing the ground beef in a petri dish (9 cm dia. x 1.5 cm thickness) to ensure patty uniformity. The study was repeated three to five times using one batch of ground beef With a fat content of 15.4i2.0%. Cooking of patties Patties were fried in a teflon—coated electric flying pan at 225°C (surface temperature) for 10 nrin per side. The temperature of the flying surface was measured using a surface temperature thermometer (Pacific Transducer Corp, Los Angeles, CA). Two patties were flied for each replication, and five replicates were analyzed for each treatment. For each replicate, four subsamples were analyzed (two unspiked for concentration and two spiked for recovery). The cooked meat patties were mixed in a blender to produce a uniform sample, and flozen at -4°C until extraction. 55 Quantification of HAAs in ground beef patties Concentrations of HAAs in the beef patties were determined by the standard addition method of Gross and Grfiter (1992). Meat samples were extracted by homogenizing 30 g cooked meat with 90 g 1N NaOH. The homogenate was divided into four equal aliquots. To determine extraction recoveries, two of the aliquots were spiked with 250 ng each of IQ, MeIQ, MeIQx, DiMeIQx and PhIP dissolved in 50 Ill methanol. Samples were mixed with Extrelut diatomaceous earth to fill an Extrelut 20 column. All four extractions were made with 40 mL dichloromethane containing 5% toluene using attached Bond Elut PRS extraction columns. The PRS cartridges were washed sequentially with 6 mL 0.1N HCL, 15 mL 40% methanol in 0.1N HCL, and 2 mL water. The HAAs were then transferred to Bond Elut C18 cartridges (100mg) with 20 mL ammonium acetate buffer solution (0.5 M, pH 8.0). The cartridges were eluted with 0.8 mL MeOHzNHaOH (9:1, v/v). The elute was evaporated to dryness, and the residue dissolved in 50 ILL methanol containing 5 ng/uL caffeine as an internal standard. Separation of the HAAs was carried out on a TSK-gel ODS80-TM column (25 cm x 4.6 mm id; Tosoh Haas, Montgomeryville, PA). A precolumn (Supelguard LC-8- DB, Supelco, Bellefonte, PA) was attached between the injector port and column to filter out unwanted compounds, and the cartridge was replaced approximately every 60 injections. The flow rate of the mobile phase was 1 ml/min. The initial ratio of acetonitrilezbufl‘er (triethylamine phosphate, 0.01 M, pH 3.2) was 8:92, which increased to 17:83 during the first 10 min. The acetonitrile concentration continued to increase until the ratio was 25:75 (10 min), then 55:45 (10 min). Over the next five min, the 56 acetonitrilezbufl'er ratio was increased to 80:20 to facilitate elution of other compounds. After 35 min, the eluting solvent was returned to its initial ratio (8:92) for 10 min to allow the column to re-equilibrate before the next injection. Samples were analyzed on a Millennium 2000 HPLC system (Millipore, Milford, MA) with a photodiode array detector (Model 991) and a scanning fluorescence detector (Model 474). The identities of the peaks were established by comparing retention times of the peaks with those of the corresponding spiked samples analyzed under the same conditions. Furthermore, UV spectral characteristics of the HPLC peaks in each sample were compared with library spectra acquired from standard HAA solutions. For each experiment, before HPLC separation of the sample extracts, four aliquots (10, 15, 20 and 25 uL) of two standard mixtures of HAAs (containing 0.5 ng/uL of each compound), the cafl‘eine internal standard (5 ng/uL), and HAA standard FEMA (5 ng/uL) were injected. Linear regression (ng compound vs peak area) was performed for individual HAAs in each mixture. A correlation coefficient of 0.99 or greater was considered acceptable for FEMA internal standards, and 0.97 or greater for the laboratory mixtures of HAAs. Each peak area corresponding to an HAA was corrected with the internal standard regression line and expressed as ng/g meat. The standard addition method of Gross and Grt'rter (1992) was then used for determining extraction efliciency and for quantification of HAAs. Each data point consisted of four subsamples; two spiked and two unspiked. The average area of the spiked samples I minus the average of the unspiked samples allowed comparison with the regression line for the standard mixture. Each data point was then corrected for its individual extraction eficiency, or percent yield. Concentrations of each HAA formed were 57 determined using the average of the two unspiked subsamples. The linear regression slope for FEMA was used used to determine the exact amount of each HAA present in each sample. Statistical analyses The results were analyzed by Sigma Stat 2.0 (Jandel Corp, San Rafael, CA). One-way analysis of variance (AN OVA) was performed for each HAA. Appropriate Comparisons were made using Student-Newman-Keuls test for one-way AN OVA analysis (Neter et al., 1993). I RESULTS AND DISCUSSION In an initial study, ground beef patties were flied at 180°C for 10 min per side. However, these cooking conditions produced little or no HAA formation in the control patties or in those containing the whole garlic or sulflrr compounds. Consequently, patties were flied at 225°C for 10 min per side to generate suflicient quantities of HAAs to evaluate the inhlbitory effects of garlic and selected organosulfur compounds on HAA formation. The dominant HAA in flied ground beef patties was PhIP, followed by MeIQx, and DiMeIQx. IQ and MeIQ are found infl'equently in cooked beef, and at very small concentrations (Skog et al., 1995). The quantification of IQ and MeIQ compounds in cooked beef is problematic because of dificulties with co—elution and peak interference (Skog et al., 1995). Average recoveries of HAAs added to the cooked ground beef patties were 74i16, 74i19, and 66i16% for MeIQx, Di MeIQx, and PhIP, 58 respectively. These recoveries are comparable to. those reported by Knize et al. (1997) I and Britt et al. (1998). Salmon et al. (1997) reported recoveries ranging flom 35 to 98% for IQ, MeIQx and DiMeIQx and from 9 to 63% for PhIP, while Britt et al. (1998) reported average recoveries of 84%, 73%, and 65% for MeIQx, Di MeIQx, and PhIP, respectively. Minced garlic, when added at levels of 4.8, 9.1, 13.0, and 16.7%, inhibited HAA formation (i.e., the sum of the concentrations of MeIQx DiMeIQx and PhIP) by 28, 59, 63, and 68%, respectively (Table 1). Analysis of variance revealed that the addition of minced garlic at levels greater than 9.1% significantly (p < 0.05) reduced the formation of HAAs in. flied ground beef patties. Statistical analysis also revealed no significant difference among the percentages of inhibition by these levels of minced garlic (Table 1). Several literature reports allude to HAA reduction by the addition of garlic and onion to meat products before cooking. Murkovic et al. (1998) reported that rosemary, thyme, sage, garlic, and brine reduced PhIP concentrations in flied ground beef by 39, 100, 64, 78, and 100%, respectively. Salmon et al. (1997) demonstrated that marinating chicken breast 20 min before grilling reduced PhIP formation by 92-99%. While the marinade contained olive oil, brown sugar, cider vinegar, lemon juice, garlic, salt, and mustard, the inhibitory effect was not assigned to any particular ingredient. Kato et al. (1998) reported that onion juice reduced mutagenicity of cooked ground beef and concluded that the sugars present in onion juice were responsible for the inhibitory action. Fresh onion contains 7.6% (w/w) sugar. On a dry matter basis, onion cOntains 33-44g (w/w) of soluble sugars (fluctose, glucose, and sucrose) per 100g dry matter. 59 Table 1. Effect of minced garlic cloves on the formation of heterocyclic aromatic amines in ground beef pattiesl Heterocyclic aromatic amines (ng/g)2 Treatment MeIQx DiMeIQx PhIP Total Inhibition (%) HAAs of total HAA formation Control 53:09“ 29:05'1 13.5i2.1‘ 21.7 5g Garlic 46:0.8' 22:04: 8.9i1.l’ 15.7 28 10g Garlic 27:06b 13:03b 50:11b 8.9 59 153 Garlic 24:06b 12:03" 44:09b 9.0 63 20g Garlic 20:08b 1.1:02b 39:10b 6.9 68 ‘ Values are based on measured cooking losses for individual patties. 2 Means with the same superscript are not significantly difl‘erent (p>0.05). Data are the means of three replicates. 60 The inhibitory effect of sugars on HAA formation has been reported by other investigators. Skog et al. (1992) demonstrated that glucose and pure lactose or lactose flom milk powder, when added to beef patties at concentrations up to 4 %, reduced mutagenicity by 34 to 76%. On the other hand, garlic has a sugar content of only 0.1% (w/w) (Lawson, 1993), and thus we conclude that the inhibitory effect of garlic is not likely to be due to the presence of sugars. The addition of DAD to ground beef patties at concentrations of 0.17, 0.34, 0.67, 0.84, and 1.01 mmol inhibited total HAA formation by 45, 68, 78, 83, and 84%, respectively (Table 2). DAD, when added at 0.67 mmol and higher, significantly (p < 0.05) reduced MeIQx, DiMeIQx and PhIP in flied ground beef patties. There was, however, no significant difference in the percent inhibition achieved by these concentrations of DAD (Table 2). At the 0.67 mmol concentration, DAD reduced the total HAA concentration in the cooked patties flom 21.2 to 4.7 ng/g meat (Table 2). These results agree with those of Tsai et al. (1996) who reported that DAD and DPD reduced the overall mutagenicity of boiled pork juice by 80 to 98%, respectively. Trompeta and O’Brien (1998) also demonstrated that selective organosulfur compounds such as glutathione, L-cysteine, L-cystine, and deoxyalliin inhibited HAA formation in a model system containing glucose, glycine, and creatinine. Based on the results of this initial study and on literature observations, several sulfur compounds (DAS, AMS, AM, DPD, cystine, and cysteine) were chosen for filrther investigation as inhibitors of HAA formation in cooked ground beef patties. The addition of DAD and DPD (0.67 mmol) to ground beef patties inhibited PhIP formation by 81% and 69%, respectively (Table 3). DAD and DPD also inhibited 61 DiMeIQx formation in flied ground beef patties by 79% and 62%, respectively. The inhibitory effects of DAD and DPD were greater than those afforded by cysteine and cystine and the other volatile sulfllr compounds that were evaluated. Similar observations were reported by Tsai et a1. (1996). Why greater inhibition was achieved with DAD and DPD than cysteine and cystine was not explained by these investigators. The volatile disulfide afforded more effective inhibition than the other compounds which may be explained by the possible scission of the disulflde bond during heating to provide sulfllydryl groups. These groups have been implicated in the Maillard reaction (Friedman and Molnar-Perl, 1990), a reaction which has been postulated to be the involved in the formation ofHAAs (lagerstad et al., 1983a). How sulfirr compounds such as DAD and DPD inhibit H'AA formation has not been investigated in any detail. However, a possible mechanism for the inhibition is through a competitive reaction between sulfur compounds and amino acids for glucose, a key component in the reactions leading to HAA formation. Mottram and Whitfield (1995) reported that sulfur compounds may directly participate in Maillard reactions leading to meat flavor production. Alliin and deoxyalliin have also been shown to react with glucose in Maillard reactions (Yu et al., 1994). Tsai et al. (1996) showed that the addition of sulfiIr compounds to a pork meat juice system reduced the formation of Maillard reaction products and this reduction was correlated with a decrease in mutagenicity. Further studies are necessary to evaluate the mechanism of the inhibition of HAA formation by organosulfur compounds. 62 Table 2. Efl‘ect of variable diallyl disulfide concentrations on the formation of heterocych aromatic amines in ground beefpatties1 Heterocyclic aromatic amines (ng/g)2 Treatment MeIQx DiMeIQx PhIP Total Inhibition (%) HAAs of total HAA formation Control 5.0i0.6’ 26:02a 13.6il.4‘ 21.2 0.17 mrnol DAD 3.6:03' 1.4:02a 67:09" 11.8 45 0.34 mmol DAD 2.5:03" 1.0:01a 33:04c 6.8 68 0.67 mmol DAD 1.7:02" 05:0.1" 25:03" 4.7 78 0.84 rnrnol DAD 16:02" 05:01" 14:02" 3.5 83 1.01 mmol DAD 15:01" 05:01" 14:02" 3.4 84 1 Values are based on measured cooking losses for individual patties. 2 Means with the same superscript are not significantly different (p>0.05). Data are the means of three replicates. 63 Table 3. Efl‘ect of various sulfur compounds on the formation of heterocyclic aromatic amines in ground beefpattiesl'2 Heterocyclic aromatic amines (ng/g)3 Treatment MeIQx DiMeIQx PhIP Control 5.23:0.9‘ 2.9:05‘ 13.5:21‘ DAD 1.7:02f 0.6:02e 26:11" Inhibition (%) 67 79 81 DAs 28:03" 13:05" 55:12“ Inhibition (%) 46 55 59 AMS 33:03" 15:05" 64:13"-c Inhibition (%) 38 49 52 AM 35:05" 1.7:05‘3" I7.3:1.5"'° Inhibition (%) 34 43 46 DPD 26:06" 11:04" 42:09“ Inhibition (%) 51 62 69 Cystine 40:06" 2.0:0.5"’° 88:13" Inhibition (%) 25 32 35 Cysteine 4.23:0.53" 2.3:05" 105:1.4" Inhibition (%) 19 22 - 22 ‘ Values are based on measured cooking losses for individual patties. 2 Sulflrr compounds were added at 0.67 mmol level to ground beefpatties. 3 Means with the same superscript are not significantly different (p>0.05). Data are the means of five replicates. 64 CHAPTER THREE Reduction of heterocyclic aromatic amine formation and mutagenicity in fried ground beef patties by organosulfur compounds ABSTRACT The effects of several organosulfur compounds on heterocyclic aromatic amine (HAA) formation in flied ground beef patties and overall mutagenicity of the patties were evaluated. Organosulfirr compounds were added directly to ground beef and thoroughly blended. Patties weighing 100g were flied in a teflon—coated electric flying pan at 225°C (surface temperature) for 10 min per side. Two patties were flied for each replication, and five replicates were analyzed for each treatment. The greatest inhibition of total HAA formation was achieved with diallyl disulflde (DAD) (78%) and dipropyl disulflde (DPD) (70%). These compounds also significantly (p < 0.05) reduced overall mutagenicity, with reductions of 75 and 65% for DAD and DPD, respectively. The addition of diallyl sulfide, allyl methyl sulfide, and allyl mercaptan also significantly (p < 0.05) reduced mutagenicity, with reductions of 56, 43, and 30% being noted, respectively. The addition of cysteine and cystine, however, did not reduce the mutagenicity of cooked meat, an observation confirmed by the relatively small reductions in HAA concentrations. These results suggested that the addition of selected sulfur compounds to ground beef may be an alternative approach to reduce HAA formation and overall mutagenicity of cooked beef patties. 65 INTRODUCTION Epidemiological studies have shown that diet and life style are closely related to human cancer (Sugimura and Sato, 1982). Many mutagens and carcinogens have been identified in foods. Recently, several foods and constituents of foods have been investigated for their inhibitory or promotional effects on carcinogenesis (Knize et al., 1999; Weisburger, 1991). Heterocyclic aromatic amines (HAAs) are produced in muscle foods during cooking, and many have been shown to be mutagenic and/or carcinogenic. These compounds have been classified into two categories, pyrolytic mutagens and thermic mutagens. Pyrolytic mutagens are formed when proteins, amino acids or proteinaceous food are heated to high temperatures (>300 °C) and are characterized by a pyridine ring with an amino group attached (Skog, 1993; Wakabayashi and Sugimura, 1998). Thermic mutagens are formed at lower temperatures (<3 00 °C) and several have been identified in cooked fish and meat products. These compounds, also called aminoirnidazoazaarenes, have been characterized as quinolines, quinoxalines, pyridines or fllropyridines. The most commonly found HAAs in foods are IQ (2-amino-3- methylimidazo[4,5-j]-quinoline); MeIQ (2-amino-3,4dimethylimidazo[4,S-f]- quinoline); MeIQx (2-amino-3,8 dimethylinridazo [4,5-j]-quinoxaline); 4,8 DiMeIQx (2-amino-3,4,8 trimethylimidazo[4,5-fl-quinoxaline); and PhIP (2-amino-1-methyl-6- phenylirnidazo [4,5-b]-pyridine) (Knize et al., 1999; Skog, 1993; Wakabayashi and Sugimru'a, 1998). 1 Garlic has been cultivated since antiquity and has been used as foodstuffs and medicines. In recent years, garlic has become highly valued because of its excellent 66 effectiveness on arteriosclerosis, its ability to lower serum cholesterol and triglyceride levels, and for its hypotensive, anticarcinogenic and antidiabetic effects. Garlic also inhibits thrombocyte aggregation and activates fibrinolysis (Kabelik, 1970; Kendler, 1987; Kritchevsky, 1991; Lutomski, 1983; Reuter and Sendl, 1994; Weiss, 1986). Food preparation methods have a significant influence on HAA formation and much research has been devoted to the carcinogens in flied and broiled food. Food preparation methods in many homes include the cooking of meats with various vegetables containing naturally occurring organosulflrr compounds. In the previous chapter, it was demonstrated that garlic and several endogenous organosulfirr compounds, when added to ground beef patties before flying, will inhibit or greatly reduce HAA formation. However, there is a need to ascertain if there is a concomitant reduction in overall mutagenicity of the flied beef with the reduced concentrations of HAAs. This need is based on the proposition that organosulfilr compounds could react with meat components during cooking to generate other mutagenic species. Therefore, the objective of this study was to characterize the relationship, if any, between the reduction of total HAA concentrations and overall mutagenicity of flied ground beef patties to which were added selected organosulfur compounds before flying. 67 MATERIALS AND METHODS Safety Heterocyclic aromatic amines are mutagenic and/or carcinogenic and should be handled with appropriate safety precautions, including the use of goggles, latex gloves and efficient fume hoods. Materials Diallyl disulfide (DAD), dipropyl disulfide (DPD), diallyl sulfide (DAS), allyl methyl sulfide (AMS), allyl mercaptan (AM), cysteine, cystine, and dimethyl sulfoxide (DMSO) were purchased from Fluka Chemical Co. (Buchs, Switzerland). The HAA standards (MeIQx, 4, 8-DiMeIQx, and PhIP) were obtained flom Toronto Research Chemicals (Toronto, Canada). The ’ HAA standard (FEMA-Flavor and Extracts Manufacturer’s Association) and the internal standard, caffeine, were gifts flom Dr. Mark Knize, Lawrence Livermore National Laboratory, Livermore, CA. The FEMA standard contained IQ, MeIQ, MeIQx, DiMeIQs, and PhIP, each at 5 ng/pL. Propyl- sulfonic acid (PRS) Bond-Elut columns (500 mg) and C18 (100 mg) cartridges were purchased flom Varian Inc. (Harbor City, CA). Extrelut-20 columns and Extrelut diatomaceous earth were obtained flom E.M. Separations Technology (Gibbstown, NJ). All other chemicals were of analytical grade and were purchased flom Fisher Scientific (Fair Lawn, NJ). Freshly ground beef was obtained flom a local supermarket and used within one hour of purchase, or stored at -20°C until required for flying. The fat content was determined by the method of F olch et a1. (1957). 68 Preparation of ground beef patties Patties were prepared flom ground beef as follows: control patties (ground beef patties mixed with 1 mL methanol) and patties containing organosulfur compounds added as separate treatments two hours before flying. Organosulfur compounds (DAD, DPD, DAS, AMS, AM, cysteine, and cystine) at a concentration of 0.67 mmol were dissolved in 1 mL methanol, added directly to the ground beef, and mixed in a Keebler mixer (Keebler Inc., Chicage, IL) for 2 min. Each patty (100g) was formed by placing the ground beefin a petri dish (9 cm dia. x 1.5 cm thickness) to ensure patty uniformity. The study was repeated three to five times using one batch of ground beef with a fat content of 15.4:2.0%. Cooking of patties Patties were flied in a teflon-coated electric flying pan at 225°C (surface temperature) for 10 min per side. The temperature of the flying was determined using a surface temperature thermometer (Pacific Transducer Corp, Los Angeles, CA). Two patties were flied for each replication, and five replicates were analyzed for each treatment. For each replicate, four sub-samples were analyzed (two unspiked for concentration and two spiked for recovery). The cooked meat patties were mixed in a blender to produce a uniform sample and flozen at -4°C until extraction. Extraction of HAAs from meat samples The HAAs were extracted flom the meat samples and purified using solid-phase chromatography following the standard addition procedure of Gross and Griiter (1992). 69 Meat samples were extracted by homogenizing 30 g cooked meat with 90 g 1N NaOH. The homogenate was divided into four equal aliquots. To determine extraction recoveries, two of the aliquots were spiked with 250 ng of each of the following HAAs (IQ, MeIQ, MeIQx, DiMeIQx and PhIP) dissolved in 50 [.11 methanol. Samples were mixed with Extrelut diatomaceous earth (V arian, Inc, Harbor city, CA) to fill an Extrelut 20 column. All four extractions were made with 40 mL dichloromethane containing 5% toluene (v/v) using attached Bond Elut PRS extraction cohlmns. One unspiked aliquot flom each meat sample was processed for the Ames/ Salmonella assay as indicated below. For HAA analyses, the PRS cartridges were washed with 6mL 0.1N HCl, 15 mL 40% methanol in 0.1N HCl, and followed by 2ml water. The HAAs were transferred to Bond Elut C18 cartridges (100mg) with 20 mL ammonium acetate buffer (0.5 M, pH8.0). The cartridges were eluted with 0.8 mL MeOHzNHaOH (9:1, v/v). The elutes were evaporated to dryness and dissolved in 50 IJI methanol containing 5 ng/ul caffeine as an internal standard. For mutagenic activity testing which does not require further sample clean-up, the remaining aliquot was ehrted flom the PRS cartridge with 2.0 ml MeOH-NI-IsOH, evaporated to dryness and dissolved in 120 uL dimethyl sulfoxide (DMSO). HPLC analyses HAA analyses were performed by high pressure liquid chromatography (HPLC) as described in Chapter 2. 70 Salmonella mutagenicity assay The mutagenic activity of the sample extracts was determined using the standard plate. incorporation assay described by Ames et al. (1975) using Salmonella ophImurium TA98 (Molecular Toxicology, Inc; Boone, NC). Aroclor-induced rat liver S-9 mixture (Molecular Toxicology, Inc) was used for metabolic activation. DMSO was used as a negative control (spontaneous revertant colonies), while 2- arninoanthracene was used as a positive control for S. whimrm'um TA98. The latter gave an average of 850 revertants/ug. To determine calculated values of revertants/ g meat, individual HAA standards (MeIQx, DiMeIQx, and PhIP) were tested under similar conditions. The concentrations of HAAs obtained by HPLC analyses were then multiplied by these values to determine the calculated overall mutagenicity. Mutagenic activity was calculated flom the linear portion ofthe dose-response curve using the method of Moore and Felton (1983). A minimum of four dose points flom duplicate platingswereused,andthelinearportionofthecru'VeswasusedtocalaIlatethe revertants/g cooked beef patties. Statistical analyses The results were analyzed by Sigma Stat 2.0 (Jandel Corp, San Rafael, CA). One-way analysis of variance (ANOVA) was performed. Appropriate comparisons were made using Student-Newman-Keuls test for one-way ANOVA analysis. Calculation of mutagenic activity was made by linear regression analysis of the dose response curves of revertants/ug of HAAs or g of cooked beef patties (Neter et al., 1993). 71 RESULTS AND DISCUSSION Reduction of HAA formation and overall mutagenicity in ground beef patties by organosulfur compounds Ground beef patties were flied at 225°C for 10 min/side to evaluate the efl‘ect of organosulfur compounds on HAA formation. Confirming what was established in Chapter 2, various organosulflrr compounds, when added directly to ground beef before flying, inhibited HAA formation to varying degrees (Table 1). The addition of DAD and DPD to ground beef patties inhibited total HAA formation by 78 and 70%, respectively. Reductions in PhIP concentrations were 82 and 73%, respectively. These compounds also inhibited DiMeIQx formation in ground beef patties by 82% (DAD) and 69.9% (DPD). The amount of inhibition of HAA formation by DAD was higher than all the other organosulfllr compounds (DPD, DAS, AMS, and AM). These results , confirm those reported in Chapter 2, while the DAD data support the results of Tsai et al. (1996) who demonstrated that DAD was an efl’ective inhibitor of mutagen formation in boiled pork juice. Several other literature reports allude to the reduction of mutagens in meat products through the addition of onion or garlic extracts. Reduction in nrutagen formation in cooked ground beef with onion juice has been reported by Kato et al. (1998). Murkovic et al. (1998) reported that garlic reduced PhIP concentrations in flied ground beef by 78%, although these investigators did not specifically address the mode of action involved, or measure the impact of HAA reduction on overall mutagenicity. The results presented here clearly confirm selected organosulfur compounds as effective 72 Table 1. Effect of various sulfur compounds on the formation of heterocych aromatic amines in ground beefpatties"2 Heterocyclic aromatic amines (ng/g)’ Treatment MeIQx DiMeIQx PhIP Total HAAs Control 6110.8" 2.8105“ 15.7:2.1‘ 24.6 DAD 20:02" 05:0.1" 29:06" 5.4 ' Inhibition (%) 68 82 82 78 DAS 3.1:0.3"'" 1.3:0.2"'" 69:09" 11.2 Inhibition (%) 50 54 56 55 AMS 36:03" 18:03" 86:14" 13.9 Inhibition (%) 38 37 45 43 AM 39:04“ 19:03" 101:1.5" 15.8 Inhibition (%) 34 34 36 36 DPD 23:06" 09:02" 42:09" 7.4 Inhibition (%) 62 70 73 70 Cystinc 44:06" 21:04" 11.4:15" 17.9 Inhibition (%) 28 26 27 27 Cysteine 50:05" 24:05" 12.8i1.8‘ 20.2 Inhibition (%) 19 14 18 17 ' Values are based on measured cooking losses for individual patties. 2 Sulfirr compounds were added at the 0.67 mmol concentration to ground beef patties. 3 Means with the same superscript are not significantly different (p>0.05). Data are the means of five replicates. 73 inhibitors of HAA formation in ground beef patties, although at the concentrations used they would probably impart an unacceptable flavor to the beefpatties. The effect of these organosulfur compounds on the overall mutagenicity of cooked beef patties was evaluated by the Ames S. whimurium assay using the tester strain TA98 (Figure 1). DAD and DPD significantly (p < 0.05) reduced mutagenicity by 75 and 65%, respectively, with the munber of revertants being lowered flom 905 revertants/g of meat to 226 and 321 revertants/g of meat, respectively. The addition of DAS, AMS, and AM also significantly (p < 0.05) reduced mutagenicity, with reductions of 56, 43, and 30%, respectively, being obtained. However, cysteine and cystine did not significantly reduce mutagenicity of the cooked meat patties. These results are comparable to those reported by Tsai et al. (1996) who demonstrated a 80-98% reduction in overall mutagenicity in boiled pork juice through the addition of DAD and DPD. They also reported no significant impact of cysteine and cystine on overall mutagenicity of the boiled pork jucie model system. Trompeta and O’Brien (1998) also demonstrated the mutagenicity-lowering activity of various sulfur compounds in a model system containing glucose, glycine, and creatinine. Measured and calculated mutagenicity in fried ground beef patties The S. typhimurium mutagenicity test is desirable when assessing the evaluation of overall mutagenicity by the reduction of HAA concentrations in meat products with specific food ingredients. There is a possibility that other mutagenic compounds may be introduced into the flied ground beef through the interaction of the potential inhibitor with meat components, or by the thermal breakdown of the inhibitor itself. The results 74 Revertantlg of Meat Control DAD DPD DAS AMS AM cystine Cysteine Figmtlflheefl’ectoforganosmfineonmoundsonflrenntaguidtyofgmmd beefpatties as determined bytheAmesS. ophinmriquA” assay. Bars with different letters are significantly different (p<0.05). All treatments were replicated five times. 75 presented hereindicatethattheinln’bition ofHAAformationinfiiedground beefpatties by organosulfur compounds, as measured by HPLC analyses, is accompanied by a concomitant reduction in the overall mutagenicity of the patties. The mutagenic activity of each HAA standard was determined by the S. whimw'ium TA 98. The mutagenic activity of MeIQx, DiMeIQx, and PhIP standard determined by the S. whimurium TA 98 were 82900, 19800, and 1900 revertants/pg of HAA, respectively. These results agree with the data ofWakabayashi and Sugimura (1998), who demonstrated that MeIQ is the most mutagenic of the HAAs evaluated, followed by IQ, MeIQx, DiMeIQx, and PhIP. Although PhIP comm less than 18% of the total mutagenic activity of meat, it is the most abundant HAA formed in cooked meat (Skog et al., 1995). Therefore, it would be expected that a significant reduction of the mutagenicity of cooked beef patties in this study would also mean that there were meaningfirl reductions in the concentrations of MeIQx and DiMeIQx, in addition to PhIP. We were interested in correlating the measured mutagenicity of the flied patties bytheS.t)phimwiumassaywithrmuagenicityvaluescalwlatedfi'omflre concentrations of the HAAs in the fried ground beef that were quantified by HPLC. TheAmesassaymaydetenninemutagerficacfivitynottotaflyaccmnnedfmbyHAA concentrations. The plot of measured and calculated mutagenicity is shown in Figure 2. Themeasured mutagenicityineach samplewas quitesimilartothennrtagenicityvalues calculated from the determined concentrations of HAAs. These observations agree with those of Felton et al. (1994) who reported that measured mutagenicity in flied beef patties was similar to the mutagenicity calculated from the measured concentrations of 76 HAAs. The linear regression between measured and calculated activities (slope 0.88, R2 = 0.96) indicates that the concentrations of the determined HAAs are responsible for most, but not all, of the mutagenicity detected. Other HAAs such as IQ, IQx, and MeIQ are found infrequently in cooked beef, but when present, are there in very low amounts. Felton and Knize (1990) reported that PhIP in flied ground beef accounted for 86-91% of the total mass of the mutagenic compounds. MeIQx and DiMeIQx contributed 9.4- 11.7% of the total mass, while IQ, IQx, and MeIQ were only found in very small concentrations. The results also demonstrated that the addition of sulfur compounds did not result in the formation of other mutagenic compounds in the ground beef through the interaction of the potential inhibitor with meat components, or by the thermal breakdown of the inhibitor itself. The scatter in the data is probably due to a combination of measurement errors in solid-phase extraction procedures and the accumulation of errors in each analytical method. Knize et al. (1994) and Turesky et al. (1988) reported that about 80% of the mutagenicity could be accounted for by quantitative HPLC analyses of HAAs in cooked beef and that the S. whimurium mutagenicity assay would be a reasonable screening method to determine HAA formation in cooked meat samples. On the basis of the results of the present study, the latter procedure could effectively evaluate the effects of selected food ingredients on HAA formation in fried ground beef patties. 77 Revertantslg of Meat (Measured) 1000 y = 0.8814: - 20.144; R2 = 0.962 800 - 600 l 400 ~ 200 « 0 T T I T T O 200 400 600 800 1000 1200 Revertantslg of Meat (Calculated) Figure 2. Plot of mutagenic activity quantitated by the S. typhirnurium TA98 assay and mutagenic activity calculated from the heterocyclic aromatic amine concentrations in fried beef patties as determined by HPLC analyses. Each data point is the mean of five replicated experiments. 78 CHAPTER FOUR A model system study of the inhibition of heterocyclic aromatic amine formation by organosulfur compounds ABSTRACT Organosulfur compounds and sodium bisulfite significantly inhibited (p<0.05) heterocyclic aromatic amine (HAA) formation in model systems containing phenylalanine, creatinine, and glucose. However, there was no inhibition by the same compounds in a model system containing only phenylalanine and creatinine. Diallyl disulfide (DAD) and dipropyl disulfide (DPD) concentrations in the model system significantly decreased (p<0.05) after heating for 10 min at 180°C. These decreases could be through their interaction with glucose and/or by their decomposition. Only very low concentrations of sulflrydryl groups (4.19 urnol and 4.00 mol) were formed on heating DAD and DPD for 30 min. Reaction of glucose and DAD produced several alliin-containing compounds. Afier 10 min ofheating at 180°C, HAA formation in the control model systems was increased significantly, while DAD was an effective inhibitor during this heating period. Tetrahydrothiophene—3-one (THT) and tetrahydrothiophene (THP); two products assessing from the interaction of glucose and DAD, had no direct influence on HAA formation in the model systems. These observations indicate that DAD and DPD may function as HAA inhibitors by their ability to react with glucose, thus possibly reducing the latter’s availability to react with phenylalanine. 79 INTRODUCTION When food is cooked, carbonyl and amino compounds react via what is known as the Maillard reaction (Hodge, 1953). Several hundreds of reaction products are produced, some of which contribute to the color and flavor of the cooked foods. The Maillard reaction may also impact the nutritional value of the food (Reynolds, 1963). Furthermore, in some cases, the Maillard reaction can lead to the formation of genotoxic compounds called heterocyclic aromatic amines (Powrie et al., 1981; Shibamoto et al., 1981; Spingarn and Garvie, 1979; Wei et al., 1981). Heterocyclic aromatic amines (HAAs) are formed in cooked meat and fish products, most likely from the reaction of proteins, sugar and creatine (Skog, 1993). These compounds have been shown also to induce colon, breast, pancreas, and prostate cancer (Stavric, 1994). The most commonly found HAAs in foods are IQ (2-amino—3- methylimidaao[4,5-j]-quinoline); MeIQ (2-amino-3,4dimethylimidazo [4,5-f]- quinoline); MeIQx (2-amino-3,8 dimethylimidazo [4,5-j]-quinoxaline); 4,8 DiMeIQx (2-amino-3,4,8 trimethylimidazo[4,5-f]-quinoxaline); and PhIP (2-amino-1-methyl-6- phenyfimidazo [4,5-b]-pyridine) (Knize et al., 1999; Skog et al., 1992; Wakabayashi and Sugimura, 1998). PhIP shows weak mutagenic activity in the Ames Salmonella assay, but is the most abundant mutagen present in cooked food (Stavric, 1994). It has been postulated that pyrazines/pyridines, aldehydes, and creatine/creatinine are condensed to form IQx- and IQ-type compounds (lagerstad et al., 1983a; Milic et al., 1993). It has also been determined that PhIP is formed from the reaction of creatinine and glucose with certain amino acids such as phenylalanine, isoleucine, or tyrosine (Johansson and lagerstad, 1993). Factors that influence the 80 formation of HAAs in foods include precursor concentrations, type of amino acids, and cooking time and temperature (Skog, 1993). Certain food ingredients can reduce HAA formation in foods such as vitamin E (Balogh et al., 2000), cherry tissue (Britt, 1998), tea polyphenolic compounds (Yen and Chen, 1995), soy protein concentrate (Wang et al., 1982) and defatted glandless cottonseed flour (Rhee et al., 1987). Garlic is a commonly used foodstuff. In addition, a variety of garlic—based health products are now readily available on the market. Most of their health promoting claims are based on the presence of organosulfur compounds such as allicin, diallyl disulfide (DAD), diallyl sulfide (DAS), and dipropyl disulfide (DPD). DAD, for example, has been shown to have a positive effect on arteriosclerosis (Kritchevsky, 1991), and on serum cholesterol (Reuter and Sendl, 1994) and triglyceride levels (Weiss, 1986). In addition, it has exhibited hypotensive (Kendler, 1987), anticarcinogenic (W attenberg et al., 1989) and antidiabetic efl‘ects (Augusti and Sheela, 1996). It has been established that organosulfur compounds will inhibit Maillard browning reactions (Friedman, 1996; Trompeta and O’Brien, 1998). The inhibition of this reaction may be the key to reducing HAA formation in foods through the addition of garlic and other organosulfur compounds. However, the mechanism by which organosulfur compounds inhibit the Maillard reaction has not been firlly elucidated. We have established that organosulfur compounds, when added to ground beef patties before flying, will inhibit or greatly reduce HAA formation and overall mutagenicity (Chapters 2 and 3). However, the mechanism by which HAA formation is influenced by organosulfirr compounds is unclear. The objective of this study is to 81 better understand how the formation of HAAs is inhibited by the addition of sulfirr compounds to food and model systems. MATERIALS AND METHODS Safety HAAs are mutagenic/carcinogenic and should be handled with appropriate safety precautions including the use of goggles, latex gloves and eflicient firme hoods. Materials Phenylalanine, glycine, glucose, creatinine, sodium bisulfite, cysteine, DTNB (5,5’-dithio—bis—(2-nitrobenzoate), tridecane, Tris-HCl, tetrahydrothiophene-3m (THT), and teetrahydrothiophene (THP) were purchased from Sigma Chemical Company (St. Louis, MO). DAD, DPD, and allyl mercaptan (AM) were purchased from Fluka Chemical Co. (Buchs, Switzerland). The HAA standards (IQx, MeIQx, and PhIP) were obtained fi'om Toronto Research Chenricals (Toronto, Canada). The HAA standard (FEMA-Flavor and Extracts Manufacturer’s Association) and the internal standard, caffeine, were gifts fi'om Dr. Mark Knize, Lawrence Livermore National Laboratory, Livermore, CA The FEMA standard contained IQ, MeIQ, MeIQx, DiMeIQs, and PhIP, each at 5 ng/uL. Propyl-sulfonic acid (PRS) Bond-Elut columns (500 mg) and C18 (100 mg) cartridges were purchased from Varian Inc. (Harbor City, CA). Extrelut-20 columns and Extrelut diatomaceous earth were obtained from BM. 82 Separations Technology (Gibbstown, NJ). All other chemicals were of analytical grade and were obtained from Fisher Scientific (F air Lawn, NJ). The heating module was a Reacti-Therm III, model 18835, made by Pierce Co. (Rockford, IL). Stainless steel test tubes, 2.3 ml capacity, with threaded, self-sealing stainless steel caps were manufactured by the Enginering Research Complex Machine Shop at Michigan State University. A new set stainless steel test tube was used for each amino acid to avoid carryover of HAAs from one experiment to another. Effect of organosulfur compounds and sodium bisnlfite on HAA formation in model systems The control model system contained 0.6 mmol phenylalanine and 0.6 mmol creatinine in 1.5 mL water, with or without 0.3 nrrnol glucose. The reactants were added directly to the stainless test tubes and sealed with threaded caps wrapped with Teflon tape. Organosulfur compounds (0.67 mrnol DAD, DPD, AM, and cysteine) and sodium bisulfite (0.67 mmol) were added to the model systems. The reaction was carried out in a closed hood separated from the rest ofthe laboratory. The Reacti- Therm heating module was allowed to preheat for a minimum of 1.5 hr before heating the samples. The heating temperature was 180° :1: 5°C and silicon oil (0.5 ml) was placed in each cavity in the heating block to facilitate heat transfer from the block to the test tubes. The stainless test tubes were heated for 30 min and then immediately cooled in an ice-bath. The contents of each test tube was quantitatively transferred to microvials (1.5 ml capacity) and stored at 5° i 1°C until required. 83 The contents of two 1.5 ml microvials were mixed with 57 ml 5N NaOH and stirred with a spatula. Aliquots (10 ml) were taken from the mixture and placed in four 250 ml beakers. The remainder of the mixture was saved and used if further extractions were required. To determine extraction recoveries, two of the aliquots were spiked with 1.0 pg each of IQx, MeIQx, and PhIP dissolved in 50 ul methanol. The rest of the extraction, purification, and quantitation procedures were similar to those described in Chapter2. Effect of heating on the stability of organosulfur compounds Organosulfur compounds (0.67 mmol DAD or DPD), with or without model system reactants (0.3 mmol glucose, 0.6 mmol phenylalanine and 0.6 mmol creatinine), were heated at 180°C for 30 min as described previously. samples were taken at 10 min intervals to quantitate DAD and DPD concentrations remaining in the model system. A HP 5890 gas chromatograph (Hewlett Packard, Avondale, PA), equipped with a fused silica capillary column (60 m x 0.25 mm id; 1 um thickness, DB-l, J&W Scientific Inc, Folsom, CA) and a flame ionization detector, was used to determine the DAD and DPD concentrations. The analytical conditions were as follows: injector temperature, 270°C; detector temperature, 300°C; helium carrier flow rate, 1 ml/min; temperature program, 40°C (5 min), 2°C/min, 260°C (60 min). A split ratio of 50:] was used. DAD and DPD concentrations were determined using the peak areas on the gas chromatograrns. The peak area of DAD or DPD from the control system before heating was standardized as 100%, and DAD or DPD concentrations from the reaction mixture were calculated. 84 Effect of heating on formation of sulfhydryl groups Each sample contained 0.67 mmol DAD or DPD in 1.5 ml water. These samples were mixed directly in the stainless test tubes and heated at 180° :t 5°C for 30 min as described previously. An aliquot (0.1 ml) of the heated sample was diluted with 0.02 ml 10 mM DTNB (5,5’-dithio-bis—(2-nitrobenzoate), 0.1 ml 0.2 M Tris-HCl bufl‘er (pH 7.5), and 0.78 ml water and its turbidity was measured at 412 nm using a UV spectrophotometer (V arian Inc, Harbor City, CA). Sulflrydryl concentrations (umoles in 1.5mL) were calculated using the Beer-Lambert law equation: AA=Aam°c-b where AA is the absorbance at 412 nm, Aam is the extinction coefficient (13,600 M1 cm’l), c is the concentration, and b is the sample thickness (1 cm). Formation of volatile compounds in heated model systems Samples containing 0.67 mmol DAD (or 0.3 mmol glucose, or 0.67 nrrnol DAD and 0.3 mmol glucose) in 1.5 ml water were heated in stainless steel test tubes at 180° i 5°C for 30 min. Afier cooling rapidly in an ice bath, the reaction mixtures were mixed with an internal standard (tridecane, 100 rd) and extracted four times with 10 ml dichloromethane. The combined extracts were dried over anhydrous sodium sulfate and concentrated to 5 ml in a Kuderna-Danish concentrator (Supelco, Bellefonte, PA). The extract was then concentrated to 1 ml under nitrogen. The generated volatile compounds were separated by gas chromatography using the same conditions as described in the previous section. 85 The concentrated isolates were analyzed by GC-mass spectrometry (MS) using a HP 5890 gas chromatograph interfaced with a HP 5970 MSD (mass selective detector) mass spectrometer. The GC/MS system was equipped with a HP 59970 Chemstation Data System. The GC was operated under the same conditions as previously described. The MS was operated in the electron impact mode with an electron energy of 70 eV and an ion source temperature of 250°C. Compounds were introduced to the ion source directly fi'om the capillary column in the GC using an open-split interface. A continuousscanmodewithascantimeofl secoveramassrangeof40-300was employed. The GC/MS data were monitored, stored and analyzed using a HP Chemstation data system. Several compounds in the isolate were identified by comparing their mass spectral data with those of authentic compounds available in the NIST/EPA/MSDC Mass Spectral Database purchased from ACS Publication Co. (Washington, DC) or INRA MassSpectra Computer Library (Laboratoire de Recherche sue les Aromes, Dijon, France). Effect of heating time on HAA formation and inhibition in model systems containing DAD or DPD Several model systems were evaluated. The control system contained 0.6 mmol phenylalanine, 0.6 nrmol creatinine, and 0.3 nrrnol glucose in 1.5 ml water. Other systems contained 0.67 mmol DAD (or DPD) in addition to the three primary reactants. Samples were heated for 30 min at 180°C as described previously, with 3 ml aliquots being taken at 10 min intervals. The effect of the sulfur compounds on HAA formation 86 was determined by extraction, purification, HPLC analysis, and HAA peak identification as described earlier. Effect of tetrahydrothiphene—3-one (THT) and tetrahydrothiophene (THP) on HAA formation in various model systems The direct effect of THT and THP on HAA formation in various model systems was evaluated by adding (0.67 mmol THT of TIH’) to model systems containing 0.6 nrrnol creatinine, 0.6 mrnol phenylalanine or glycine, and with or without 0.3 mrnol glucose in 1.5 ml water. The reactants were heated at 180°C for 30 min. Samples were extracted and purified aspreviously described and analyzed byHPLC. Statistical Analysis The results were analyzed by Sigma Stat 2.0 (Jandel Corp., San Rafael, CA). One way analysis of variance (ANOVA) was performed for each HAA Appropriate comparisons were made using Student-Newman—Keuls test for one way ANOVA analysis. 87 RESULTS AND DISCUSSION Effect of organosulfur compounds and sodium bisulfite on HAA formation in model systems The major HAAs detected in the heated model systems containing glucose, phenylalanine, and creatinine were IQx, MeIQx, and PhIP (Table 1). As expected, the dominant HAA was PhIP (34.4i8.37 nmol/mmol creatinine), followed by MeIQx (12.91698 nmol/mmol creatinine) and IQx (7.2i4.17 nmol/mmol creatinine). These observations agree with those of Shioya et al. (1987) and Skog and lagerstad (1991). The average recoveries of HAAs from the model system were 87:1:16, 85i18, and 82:15% for IQx, MeIQx, and PhIP from similar model systems, respectively. These recoveries are comparable to those of Scranton (1997) who reported average recoveries of 88, 88, and 71% for IQx, MeIQx, and PhIP, respectively. Phenylalanine is the major anrino acid contributing to PhIP formation in meat and model systems (Shioya et al. 1987; Skog and lagerstad, 1991). Concentrations of PhIP in beef have been estimated to be ten times greater than those of the other known HAAs combined (Felton et al., 1986b). Therefore, it may play an important role in the etiology of cancer, even though PhIP is less mutagenic than other HAAs (Stavric, 1994). Concentrations of PhIP were significantly reduced (p < 0.05) upon the addition of organosulfirr compounds to the model systems before heating. The percentage inhibition ranged from 73% for DAD to 42% for AM. MeIQx concentrations were reduced 82% by DAD and 63% by DPD. Cysteine did not significantly reduce HAA 88 up .' .0 Table 1. Efl‘ect of organosulfur compounds and sodium bisulfite on the formation of heterocyclic aromatic amines in a model system containing phenylalanine, creatinine, and glucose1 Heterocyclic aromatic amines (nmol/mmol of creatinine) Treatment IQx MeIQx PhIP Control 72142“ 12913.0“ 34418.4" DAD 1.3111" 2210.7c 3214.2c Inhibition (%) 75 82 73 DPD 2911.8" 4911.9" 12.6137c Inhibition (%) 61 63 56 Bisulfite 1,710.4" 2,110.7c 7,812.0c Inhibition (%) 77 84 78 AM 4.411 .5a 7.5122“ 19.9141" Inhibition (%) 39 42 42 Cysteine 5.612.2‘ 10213.4a 29.516.5‘ Inhibition (%) 22 20 14 “Means with different superscripts are significantly different (p<0.05). Comparisons are made only within the same column. Meansistandard deviations; n=5 for all treatments. lHeated at 180°C for 30 min. 89 formation, an observation similar to that described previously for fiied ground beef patties (Chapter 2). Sodium bisulfite also significantly (p < 0.05) inhibited HAA formation, with reductions of 77, 84, and 78% being achieved for IQx, MeIQx, and PhIP, respectively. The inhibitory effect of sodium bisulfite on HAA formation has been reported previously by Krone and Iwaoka (1987), who observed a reduction in the mutagenicity of canned salmon upon the addition of bisulfite. Chen (1988) also demonstrated sodium bisulfite inhibition of IQx, MeIQx and DiMeIQx formation in fiied ground beef patties, although he did not ofl‘er an explanation for its inhibitory action. The inhibition of the Maillard reaction by sodium bisulfite has been established through its interaction with reducing sugars (W edzicha, 1992). Several chemical mechanisms have been proposed to explain its inhibitory action One ofthe most important involves its addition to the carbonyl group of reducing sugars and other carbonyl compounds participating in the Maillard reaction (W edzicha, 1992). The reaction between sodium bisulfite and the carbonyl group of reducing sugars produces a 3,4—dideoxy-4-sulfohexosulose, a result of nucleoplrilic attack by the sulfite ion on the a,B-unsaturated carbonyl moiety of 3,4-dideoxyhexosulos—3-ene. This reaction may compete with pathways that are involved in further Maillard reactions (W edzicha, 1992) It has been speculated that HAA formation occurs through intermediates of the Maillard reaction. lagerstad et al. (1983a) proposed that pyridines and pyrazines, formed via the Maillard reaction, react with an aldehyde to form a quinoline or quinoxaline. Such structures are integral parts of the HAA molecule. Creatine undergoes dehydration and cyclization to form creatinine when heated, which then reacts with an aldehyde to form an IQ- or IQx-type HAA. Inhibition of the Maillard reaction by sodium bisulfite thus appears to be a likely mechanism by which HAA formation is inhibited/reduced in those systems containing bisulflte. To further establish that the inhrbitory action of sodium bisulfite, and possibly that of the organosulfur compounds under evaluation, is via reaction with glucose, a study was designed to investigate HAA formation in model systems containing phenylalanine and creatinine (i.e., sugar-free). The only HAA detected in this system was PhIP (Table 2). These results agree with those of Overvik et al. (1989) and Skog and lagerstad (1991). The latter investigators fiIrther demonstrated that the yield of PhIP increased three-fold when glucose was added to the reaction mixture and heated under similar conditions. Furthermore, small concentrations of the IQ-type HAAs were present in the model systems. Our results (Tables 1 and 2) confirmed these observations, both with respect to the effect of glucose on HAA formation (3-4 fold increases in PhIP concentrations) and to the quantities of the other HAAs produced. Sodium bisulfite and organosulfur compounds did not inhibit PhIP formation in the model systems that did not contain glucose (Table 2). Because sodium bisulfite reacts directly with glucose, it may be inferred that HAA inhibition by DAD and DPD in model systems containing glucose could also be through their interaction with glucose. Understanding how this occurs is the basis of the next series of experiments. Effect of heating on the stability of organosulfur compounds 91 Table 2. Effect of organosulfirr compounds and sodium bisulfite on the formation of heterocyclic aromatic amines in a model system containing phenylalanine and creatininel Heterocyclic aromatic amines (nmol/mrnol of creatinine) Treatment IQx MeIQx PhIP Control ND ND 10.212.2' DAD ND ND 10.511 .5' DPD ND ND 1 1.111 .7‘ Bisulfite ND ND 10.211 .7‘I 1'4’Means with different superscripts are significantly difl‘erent (p<0.05). Comparisons are made only within the same column. Means15tandard deviations; n=5 for all treatments. ND = Not detectable. Limit of detection is 0.4 ng. lHeated at 180°C for 30 min. 92 In order to determine whether DAD or DPD undergoes decomposition in the model systems, a study was designed to quantitate DAD or DPD concentrations afier 10, 20, and 30 min of heating at 180°C. Figure 1 shows that DAD and DPD concentrations in the model system containing glucose, creatinine, and phenylalanine decreased by 57 and 46%, respectively, after only 10 nrin of heating at 180°C. These decreases may be due to their interaction with components of the modelsystem and/or by their own thermal decomposition. When DAD (or DPD) was heated alone under similar conditions (30 nrin at 180°C), markedly smaller decreases in concentration (24- 43%) were observed (Figure 1). These decreases may be attributed to decomposition of the sulfur compounds or by their vaporization fi'om the sealed tubes. The difl‘erences in the percentage losses of DAD, and DPD in the various model systems may be explained by their interaction with other model system reagents, namely glucose, creatinine, and phenylalanine. A possible mode of loss of the disulfide compounds is through dissociation to sulfhydryl compounds on heating. This time of inquiry is important because Friedman and Molnar-Perl (1990) proposed that sulflrydryl groups inhibit the Maillard reaction through their interaction with intermediates formed during the Maillard reaction and by suppression of flee-radical formation. Heating DAD and DPD at 180°C for 30 min produced low concentrations of sulfhydryl compounds, 4.19 and 4.00 umol being produced fi'om heating DAD and DPD, respectively (Figure 2). These numbers represent conversion of 0.63% of DAD and 0.60% of DPD to their respective thiols. Thus, the sulflrydryl involvement in the inhibition of HAA formation is unlikely. It is 93 Dlalh'l disulflde or dipropyl disulflde content (%) 100 E + Phenylalnine+glucose+creatini1e+DPD son ‘ -O—Phenylalnine+glucose+creatinine+DAD +DAD 80+ —v—DPD 704 \ 30‘ "\ w-r O 40.. 30.. 20.. O 104 O o . . . o 10 20 30 Time (min) Figure LEfl‘ectofhsaingat l80°Cfor30minontheconcentrationsoforganosulfirr compmmdsmamodelsystemcontainingphenylalanme,aeafinme,andglucose. Alltreatmentsaremade intriplieate. 94 A “: r-i ET... SH content (.1 males in 1.5 ml) 4.5 + DAD 4_o - —O— DPD . shun-we, 3.5 ~ 3.0 ~ 2.5 n 2.0 - 1.5 - 1.0 - 0.5 - 0.0 I 1 Time (min) Figure 2. Sulflrydryl content on heating DAD and DPD at 180 °C for 30 min. All treatments were made in triplicate. 95 more likely that HAA inhibition by the organosulfin compounds occurs by their direct interaction with glucose. Formation of volatile compounds in heated model systems In order to determine whether the reaction between DAD and glucose could produce compounds with the potential to inhibit HAA formation, a study was designed to identify some of the principal volatile compounds produced on heating glucose, DAD, and glucose and DAD together, at 180°C for 30 min. Volatile compounds tentatively identified by mass spectrometry included methyl 2-firroate, 5- (hydroxymethyl) 2-fi1rancarboxaldehyde, 2-furancarboxaldehyde, and 1,3-dihydroxy 2- propanone (Table 3, Appendix I). These results generally agree with those of Tai and Ho (1998) and Yu et al. (1994) who reported that 2-firrancarboxaldehyde was the major thermal degradation product of glucose. The furfural group is mainly derived via sugar cararnelization. Volatile compounds identified from the thermal degradation of DAD are shown in Table 4 and include diallylsulfide, 3-vinyl-1,2-dithiocyclohex-5-ene, diallyltrisulfide, 3—(2,3-dithia-5-hexenyl)—3,4—dihydro-2H—thiopyran, 6-methyl-4,5,8,9- . tetrathiadodeca—l,11-diene, and 3-(2,3,4,trithia-6-heptenyl)—3,4-dihydro-2H-thiopyran. Block et al. (1988) identified several sulfur-containing compounds on heating DAD at 80°C for 2-10 days including thioacrolein dimer 3-vinyl-4H-[1,2]-dithin, 2-vinyl-4-H- [1,3]-dithin, diallyl sulfide, diallyl tetrasulfide, 6-metlryl-4,5,8,9-tetrathiadodeca-l,11- diene, a mixture of 2- and 3-(2’,3’-dithia-5’-hexenyl)-3,4-dihydro-2H—thiopyran, and 4, 5,9, 10-tetrathiatrideca-1, 12-diene. 96 all. 3".7-1-1‘314- fl. ... . Table 3. Compounds tentatively identified on heating glucose at 180°C for 30 min Peak CompoundsI ‘ RT (min)2 MW3 1 5-(hydroxymethyl) 2-fi1rancarboxaldehyde 16.7 126 2 2-firrancarboxaldehyde 23.3 96 3 1,3-dihydroxy 2-propanone 31.1 90 4 Methyl 2-furoate 53.4 126 1Identification of the volatile compounds was based on GC/MS analysis. 2Retention time. 3Molecular weight. 97 a. If "i Table 4. Compounds tentatively identified on heating diallyldisulfide at 180°C for 30 min Peak CompoundsI RT (min)2 MW3 1 Diallyldisulfide 16.8 146 2 Diallylsulfide 28.1 1 14 3 3-vinyl-1,2-dithiocyclohex-5-ene 39.3 144 4 Diallyltrisulfide 44.3 178 5 3-(2,3-dithia-5-hexenyl)-3,4—dihydro-2H-thiopyran 48.7 218 6 6-methy1-4,5,8,9-tetrathiadodeca-1,1 l-diene 55.6 252 7 3-(2,3,4-trithia-6-hepteny1)—3,4-dihydro-2H- 58.7 250 thiopyran 'Identification of the volatile compounds was based on GC/MS analysis. 2Retention time. 3"Molecular weight. 98 Yu et al. (1994) also identified a number of volatile compounds from the thermal degradation of alliin which is the predominant amino acid derivative of garlic and parent compound of DAD. Allyl alcohol was the predominant volatile compound, while others included acetaldehyde, allyl alcohol, acetic acid, thiazole, 2- methylthiazole, dipropyl sulfide, 3-methylthiacyclopentane, 2-formylthiophene, 3- formylthiophene, 2-methyl-1,3-dithiane, 3,6-dimethyl-1,4-dithiane, 4-methyl-1,2- dithiepane,1,2,3-trithiacyclohexane, 1,2,3,4-tetrathiepane, 4,6—dimethyl-1,2,5- trithiepane; and 4~ethyl-6-methyl-1,2,3,5-tetrathiane. Compounds identified from the heating of glucose and DAD together at 180°C for 30 min are listed in Table 5. The major compounds were tetrahydrothiophene (THP), S-methyl-Z-thiophene carboxaldehyde, and tetrahydrothiophene-3m (THT). Thiophene and thiophene-3 -one formation from the reaction of glucose and DAD can beexplainedbytheexchangeofSandOinthefirranringdmingheating(Shibamotoet al., 1981). As indicated previously, furan ring products such as 2-furancarboxaldehyde, methyl 2-furoate, and 5-(hydroxymethyl) 2-furancarboxaldehyde are thermal degradation products of glucose. A comparison of the respective GC results (summarized in Table 5 and Appendix 1) reveals the formation ofcompounds assessing fi'om the interaction of glucose and DAD. Because glucose is viewed as a major contributor to HAA formation, it is possible that the reaction of DAD with glucose reduces the availability of glucose to participate in the Maillard reaction, i.e., the carbonylamino reaction. To. further verify this hypothesis, the effect of DAD and DPD on HAA formation in a model system containing phenylalanine, creatinine, and glucose and heated at 180°C for 10, 20 or 30 min, was investigated. 99 Table 5. Compounds tentatively identified on heating glucose and diallyldisulfide at 180°C for 30 min ' Compoundsl RT (min)2 MW3 Compounds generated on heating glucose 5-(hydroxymethyl) 2-filrancarboxaldehyde 16.7 126 2-firrancarboxaldehyde 23.3 96 1,3-dihydroxy Z-propanone 31.1 90 Methyl 2-furoate 53 .4 126 Compounds generated on heating diallyldisulfide Diallyldrsulfi' de 16.8 146 Diallylsulfide 28.1 1 14 3-vinyl- 1,2-dithiocyclohex-5-ene 39.3 144 Diallyltrisulfide 44.3 178 3—(2,3-dithia-5-hexenyl)-3,4-dihydro-2H— 48.7 218 thiopyran 6-methyl-4,5,8,9-tetratl1iadodeca-l ,1 l-diene 55.6 252 3-(2,3,4-trithia-6-heptenyl)-3,4—dihydro—2H— 58.7 250 thiopyran Peak Compounds generated on heating glucose and diallyldisulfide 1 Tetrahydrothiophene (THP) 26.5 88 2 3,5-diethyl-1,2,4-trithiolane , 44.7 ' 180 3 3-(allylthio)-propionic acid 50.4 146 4 Tetrahydrothiophene-3-one (THT) 52.3 102 5 S-methyl-Z-thiophene carboxaldehyde 81.4 126 6 9-thianoradarnantane 83. l 140 1Identification of the volatile compounds was based on GC/MS analysis. 2Retention time. 3Molecular weight. 100 Effect of heating time on HAA formation and inhibition of HAA formation in model systems containing DAD and DPD In order to gain further insight into the inhibition of HAA formation by organosulfur compounds, a study was designed to determine the effect of heating time on HAA formation and inhibition by DAD and DPD in model systems containing phenylalanine, creatinine, and glucose (Figure 3). When HAA formation was evaluated over the entire heating period (30 min), approximately 63% of the HAAs were produced in the initial 10 nrin of heating. This observation could be explained by the very fast and eflicient heat transfer through the wall of the test tube and by the relatively low activation energies (689-1344 kJ/mol) of HAA formation. These results generally agree with those of Arvidson et al. (1997) and, Trompeta and O’Brien (1998) who demonstrated a rapid depletion of glucose in the early stages of the reaction involving phenylalanine, glucose and creatinine. These investigators concluded that glucose was a limiting precursor and actively participated in the formation of HAAs. The retention of creatinine and amino acids was >20%, even after 15 min ofheating, while all glucose had reacted after 2.5 nrin. Chen and Meng (1999) also observed rapid formation of HAAswithinthefirstSto10minofheatingamodelsystemcontainingglucose, phenylalanine, and creatinine at 150°C and 200°C. After this time, only a steady increase in HAA formation was observed. They concluded that this occurred possibly through the rapid exhaustion of all the HAA precursors in the reaction system. When DAD was added to the system, HAA formation over the first 10 min was significantly (p<0.05) decreased (43% reduction). When the model system was heated for 20 min, 101 ‘1 fi .4‘ u Total HAA formation (nrnollrnrnol of creatinine) 8 B .5 O 1 _Cordl'ol a Figue3. Inhibition oftotalHAAforrmtion by DADmdDPDinamodel system containing phenylalanine, creatinine, lard glucose heated at 180"C for 30 um Bars with different letters are significantly different (p<0.05). Comparisons are made only within the same column. n=3 for all treatment. 102 - .1 ?—-..~'~ A the HAA concentration was approximately 81% of that produced after 30 nrin of heating. However, the HAA concentrations formed after 10 and 20 min were not significantly difl‘erent. The addition of DAD and DPD to the model system reduced HAA formation by 60% and 51%, respectively. These results and those of previous studies suggest that HAA inhibition by DAD appears to be through its active interaction with glucose during the first 10 min of heating at 180°C. Effect of tetrahydrothiophene-Hue (THT) and tetrahydrothiophene (THP) on HAA formation in various model systems It has been postulated by Tsai et al. (1996) that THT might play an important role in HAA formation. Because of the possible implications of THT or THP as possible inhibitors of HAA formation, the next set of studies investigated their possible role in HAA formation in model systems containing phenylalanine, creatinine and glucose, and glycine, creatinine and glucose. The principal HAAs formed in the model systems containing glucose, phenylalanine, and creatinine were PhIP, IQx, and MeIQx, whereas PhIP was the only HAA produced in the control model system containing phenylalnine and creatinine (Tables6and7). TheseresultsconfirmourpreviousdataandalsoshowthatTHTand THP had no efl‘ect on HAA formation in the model systems, regardless of whether glucose was present or not. The major HAAs in the model systems containing glycine, creatinine, and glucose were IQx and MeIQx (Table 8). Scranton (1997) also reported that IQx and MeIQx were the dominant HAAs formed in the same model system under similar heating conditions. The addition of DAD significantly (p<0.05) inhibited IQx 103 Table 6. Effect of tetrahydrothiophene-3 -one (THT) and tetrahydrothiophene (THP) on formation of heterocyclic aromatic amines in a model system containing phenylalanine, creatinine, and glucose' Heterocyclic aromatic amines (nmol/mmol of creatinine) Treatment IQx MeIQx PhIP Control 6.712.4‘ 14.615.4‘ 30.918.7'I THT 6.1121" 15.7149“ 31.1191" THP 7 012.4‘ 16.014.4‘ 33.218.5'I ‘Means with different superscripts are significantly different (p<0.05). Comparisons are made only within the same column. Means1standard deviations; n=3 for all treatments. ‘Heated at 180°C for 30 min. 104 Table 7. Effect of tetrahydrotlriophene-3-one (TI-IT) and tetrahydrothiophene (THP) on formation of heterocyclic aromatic amines in a model system containing phenylalanine and creatinine1 Heterocyclic aromatic amines (nmol/mmol of creatinine) Treatment IQx MeIQx PhIP Control ND ND 11.412.7‘ THT ND ND ’ 10.212.1' THP ND ND 12.812.9' 'Means with different superscripts are significantly difl'erent (p<0.05). Comparisons are made only within the same column Means1standard deviations; n=3 for all treatments. ND= Not detectable. Limit of detection is 0.4 ng. "Heated at 180°C for 30 min. 105 Table 8. Effect of tetrahydrothiophene-3 -one (THT) and tetrahydrothiophene (THP) on formation of heterocyclic aromatic amines in a model system containing glycine, creatinine, and glucose1 Heterocyclic aromatic amines (nmol/mmol of creatinine) Treatment IQx ' MeIQx Control 8.511.9‘ 18.314.3‘ - DAD 2,310.7" 5,511.7" THT 7,112.3" 17.4139" 3 THP 3.712.7‘ 17.614.5‘ ’Means with different superscripts are significantly different (p<0.05). Comparisonsarenradeonlywithinthesamecolunm. Meansistandard deviations; n=3 for all treatments. lHeated at 180°C for 30 nrin. 106 and MeIQx formation, while THT and THP had no effect on HAA formation. Tsai et al. (1996) postulated that THT might play an important role in IQ-mutagen formation in the reflux boiling ofpork juice extracts. They concluded that reductions in the concentrations of THT and the four major Maillard reaction products (pyridines, pyrazines, thiophenes, thiazoles) correlated with a reduction in mutagenicity, even though there was no correlation with mutagenicity when each Maillard reaction product was examined alone. However, our data indicate that THT and THP are merely reaction products between glucose and DAD, and do not influence HAA formation. This divergence of opinion is probably due to the different model systems used and misleading conclusions of Tsai et al. (1996) fi'om insuficient supporting data. They proposed the possible involvement of THT in IQ-mutagen formation in boiled pork juicewithoutstudyingthespecificroleofTI-lTonHAAformation. Theresultsofour study confirm that THT and THP are merely reaction products and are not involved in HAA formation. S Y AND CONCL 81 NS The results of these studies can be summarized as follows: 1. Selected volatile organosulfur compounds and sodium bisulflte are effective . inhibitors of HAA formation in model systems containing phenylalanhe, glucose, and creatinine. 2. These compounds are not effective inhibitors of HAA formation in model systems that do not contain glucose. 107 , tan—r-r—q 3. Disulfides react directly withglucosetoproduceamlmberofsulfirr- containing compounds. 4. Such compounds (i.e., THP and THT) have no influence on the formation of HAAsinthemodelsystenm. While these experiments point to a competitive reaction between organosulfur compounds and amino acids for glucose, the mechanism by which these compounds inhibit HAA formation is still not clarified. However, the observation that DAD has no effect on HAA formation in model systems without glucose provides supporting evidencethatthehueracfionofDADwithglucoseisapossiblekeydememmits inhibition of HAA formation. It is also apparent that the products of interaction of glucose and DAD are not directly involved in the inhibition reaction. 108 CHAPTER FIVE SUMMARY AND CONCLUSIONS A series of studies were conducted to investigate the effect of sulfilr compounds on HAA formation in meat and model systems, with the overall goal of a fuller understanding of how organosulfilr compounds inhibit HAA formation. The reaction between sugar, amino acids, and creatinine in meats leads to the formation of carcinogenic/mutagenic HAAs. These compounds generally are formed flom pyrazines and pyridines that are produced by the Maillard reaction. However, as determined in this study, the formation of HAAs in meat and model systems were inhibited by the presence of organosulfirr compounds. The effects of selected organosulfur compounds (DAD, DPD, DAS, AMS, AM, cystine, and cysteine) on HAA formation in flied ground beef were studied. Organosulfur compounds were added directly to 100g ground beef and flied at 225°C for 10 nrin per side. The HAAs were extracted and purified. using solid-phase extraction, and analyzed by HPLC. The identities of the peaks were established by comparing their retention times in UV chromatograms with . those of standard references. The inhibitory effects of DAD and DPD were greater than those afl‘orded by cysteine and cystine and the other volatile sulfirr compounds that were evaluated. The addition of DAD and DPD to ground beef patties inhibited PhIP formation by 81% and 69%, respectively. DAD and DPD also inhibited DiMeIQx formation in flied ground beef patties by 79% and 62%, respectively. This study clearly demonstrated 109 . ~ _‘r."—‘i_‘7'i‘l IR. that sulfur compounds may represent an alternative approach to reducing HAA formation in cooked beef patties. ' The effects of sulfur compounds on HAA formation and overall mutagenicity of flied ground beef patties, as determined by the Ames S. {whimurium assay, was studied. Reduction of overall mutagenicity was related to the decrease in HAA formation in flied ground beef patties. The addition of DAD and DPD to ground beef patties inhibited total HAA formation by 78% and 70%, respectively. Again, inhibition of HAA formation by DAD was higher than that of all the other organosulfur compounds (DPD, DAS, AMS, AM, cysteine, and cystine). DAD and DPD significantly (p < 0.05) reduced overall mutagenicity by 75% and 65%, respectively, the number of revertants being lowered from 905 revertants/g of meat to 226 and 321 revertants/g of meat, respectively. The addition or DAS, AMS, and AM also significantly (p < 0.05) reduced mutagenicity, with reductions of 56%, 43%, and 30%, respectively, while cysteine and cystine did not significantly reduce mutagenicity of the cooked meat patties. The measured mutagenicity in each sample was quite similar to the mutagenicity values calculated flom the determined concentrations of HAAs. This study demonstrated that the addition of sulfur compounds did not result in the formation of other mutagenic compounds in the ground beef through the interaction of the potential inhibitor with meat components, or by the thermal breakdown of the inhibitor itself. A third study was designed to investigate more firlly how organosulfur compounds inhibit HAA formation in meat and model system. Addition of glucose to a model system containing phenylalanine and creatinine increased concentrations of PhIP 110 by 3-4 fold and also increased the formation of other HAAs. These studies established glucose as a major contributor to HAA formation. Volatile organosulfirr compounds and sodium bisulfite were effective inhibitors of HAA formation in model systems containing phenylalanine, glucose, and creatinine. However, these compounds did not affect HAA formation in model systems that did not contain glucose. Because the inhibition of the Maillard reaction by sodium bisulflte has been established through its direct interaction with reducing sugars, it can be inferred that a possible mechanism of HAA inhibition by DAD and DPD in model systems containing glucose could be through their interaction with glucose. DAD and DPD concentrations in the model systems containing glucose, creatinine, and phenylalanine decreased by 57 and 46%, even after only 10 min of heating at 180°C. These decreases may be explained by the ' interaction of the sulfilr compounds with components of the model system and/or by their own thermal degradation. A number of sulfur-containing compounds such as THT and THP were produced on heating glucose and DAD at 180°C for 30min HAA formation increased rapidly during the first 10 rrrin of heating, followed by. a slower increase. HAA inhibition by DAD is possibly through its active interaction with glucose during this same heating period. The products of DAD and glucose interaction, THT and THP, had no efl‘ect on HAA formation in the various model systems, regardless of whether glucose was present or not. It was concluded that THT and THP are merely reaction products between glucose and DAD and have no influence on HAA formation. While these experiments point to a competitive reaction between organosulfur compounds and anrino acids for glucose, the mechanism by which these compounds 111 inhlbit HAA formation is still not clarified. However, the observation that DAD does not influence on HAA formation in model systems that do not contain glucose provides supporting evidence that the interaction of DAD with glucose is a possible key element in its inhibition of HAA formation. It is also apparent that the products of interaction of glucose and DAD are not directly involved in the inhibition reaction. 112 CHAPTER SIX FUTURE RESEARCH The effect of organosulfur compounds on HAA formation in meat and model ‘ systems and overall mutagenicity in flied beef patties was studied. Results indicate that the addition of sulfur compounds to prior to cooking may represent an alternative approach to reduce HAA formation in cooked beef patties. However, this study revealed other questions that require some further address. 1. The role of organosulfur compounds in inhibiting HAA formation in cooked meat system has been established. However, sensory studies must be carried out to determine an acceptable level of garlic added to ground beef. Similar studies should be done with the individual organosulfur I compounds that have demonstrated an ability to reduce HAA formation in cooked meat products. It is well established that DAD and glucose produce several thermal reaction products, such as THT and THP. Studies are needed to determine how sulfur compounds interact with glucose. Using 35S-labelled DAD or 14C-labelled ‘ glucose to produce thermal reaction products may be an alternative approach to further evaluate/confirm the mechanism of DAD inhibition of HAA formation. . This study focused on the principal sulfilr compounds present in garlic. Additional investigations should be carried out with other sulfur-containing compounds such as glutathione, glutathionesulfonic acid, cysteine, cysteic acid, and cysteinesulfinic acid. Such studies would enable us to more fully 113 understand why certain sulfur compounds in food afford greater inhibition of HAA formation than do others. 111‘ 4 '.AH 114 REFERENCES Abdulkarinr, B.G., and Smith, 18. 1998. Heterocyclic amines in flesh and processed meat products. J. Agric. Food Chem. 46:4680-4687. Alink, G., Knize, M., Shen, N., Hesse, S. and Felton, J. 1988. Mutagenicity of food pallets flom human diets in the Netherlands. Mutation Res. 206:387-393. Aoyama, T., Gelboirr, H., and Gonzalez, F. 1990. Mutagenic activation of 2-arnino-3- methyl-imidazo[4,5-j]quinoline by complementary DNA-expressed human liver P450. Cancer Res. 50:2060—2063. Ames, B.N., McCann, J., and Yamasaki, E. 1975. Methods for detecting carcinogens and mutagens with Salmonella/mammalian-microsomal mutagenicity test. Mutation Res. 31 :347-364. Arvidsson, P., Van Boekel, M.A.J.S., Skog, K., and lagerstad, M. 1997. Kinetics of formation of polar heterocyclic amines in a meat model system. J. Food Sci. 62:911-916. ’ ' Arvidsson, P., Van Boekel, M.A.J.S., Skog, K., Solyakov, A., and JCDgerstad, M. 1999. Formation of heterocyclic amines in a meat juice model system. J. Food Sci. 64:216-221. Augusti, K., and Sheela, C. 1996. Antiperoxide effect of S-alkylcysteine sulfoxide, an insulin secretagogue, in diabetic rats. Experientia. 52:115-120. Balogh, Z., Gray, 11., Gomaa, EA, and Booren, AM. 2000. Formation and inhibition of heterocyclic aromatic amines in flied ground beef patties. Food Chem. Toxicol. 38:395-401. ' Barnes, W.S., Maher, IO, and Weisburger, J.H. 1983. High-pressure liquid chromatographic method for the analysis of 2-amino-3-methyl[4,5-f]imidazo- quinoline, a mutagen formed during the cooking of food. J. Agric. Food Chem. 31:883-886. Becher, G., Knize, M.G., Nes, F1, and Felton, IS. 1988. Isolation and identification of mutagens from a flied Norwegian meat product. Carcinogenesis 92247-253. Bjeldanes, L.F., Morris, M.M., Felton, J.S., Stuermer, D., Berry, P., Timourian, H, and Hatch, F.T. 1982. Mutagens flom the cooking of food 11. Survey by Ames Salmoner test of mutagen formation in the major protein-rich foods of the American diet. Food Chem. Toxicol. 20:357-363. 115 rgnien‘m ks; Bjeldanes, L.F., Morris, M.M., Timourian, H., and Hatch, PT. 1983. Effects of meat composition and cooking conditions on mutagen formation in flied ground beef. J. Agric. Food Chem. 31:18-21. Block, E., Lyer, R., Grisoni, S., Saha, C., Belman, S., and Lossing, F. 1988. Lipoxygenase inhibitors from the essential oil of garlic. Markovnikov addition of the allyldithio radical to oleflns. J. Am. Chem. Soc. 110:7813-7827. Block, E. Naganathan, S., Putnam, D., and Zhao, S. 1992. Allium chemistry. J. Agric. Food Chem. 40:2418-2430. Block, E., Naganathan, S., Putnam, D., and Zhao, S. 1993. Organosulfirr chemistry of garlic and onion: recent results. Pure Appl. Chem. 65:625-632. Block, E., Birringer, M., Jiang, W., Nakahodo, T., Thompson, H., Toscano, P., Uzar, H, Zhang, X., and Zhu, Z. 2001. Allium chemistry: Synthesis, natural occurance, biological activity, and chemistry of se-alk(en)ylselenocysteines and their 1— glutarnyl derivatives and oxidation products. J. Agric. Food Chem. 49:458—470. Botting, K.J., Young, M.M., Pearson, A.E., Harris, P.J., and Ferguson, LR. 1999. Antimutagens in food plants eaten by Polynesians: Micronutrients, phytochenricals and protection against bacterial mutagenicity of the heterocych amine 2-an1ino-3-methylirnidazo(4,5;f)quinoline. Food Chem. Toxicol. 37:95- 103. Bordia, A., Velma, S., and Srivastava, K. 1998. Effect of garlic (Allium sativum) on blood lipids, blood sugar, fibringen and fibrinolytic activity in patients with coronary artery disease. Prostaglandins Leukotrienes and Essential Fatty Acids. 58:257-263. Britt, C., Gomaa, E.A, Gray, J.I., Booren, AM. 1998. Influence of cherry tissue on lipid oxidation and heterocyclic aromatic amine formation in ground beef patties. J. Agric. Food Chem. 46:4891—4897. Butler, M., Iwasaki, M., Guengerich, F., and Kadlubar, F. 1989. Human cytochrome P- 450 (P4501A2), the phenacetin O-deethylase, is primarily responsible for the hepatic 3-dimethylation of caffeine and N-oxidation of carcinogecic arylamines. Proceedings of the National Academy of Science of the USA 86:7696-7700. Cavallito, C., and Bailey, J. 1944. Allicirr, the antibacterial principle of Allium sativium. 1. Isolation, physical properties, and antibacterial action. J. Am. Chem. Soc. 66:1950-1951. Cavallito, C., Bailey, J ., and Buck, J. 1945. The antibacterial principle of Allium sativium. 1944. HI. Its precursor and “essential oil of garlic” J. Am. Chem. Soc. 67:1032-1033. 116 Cavallito, C., Buck, J ., and Suter, C. 1944. Allicin, the antibacterial principle of Allium sativium. H. Determination of the chemical structure. J. Am. Chem. Soc. 66:1952- 1954. Chen, C. 1988. Factors influencing mutagen formation during flying of ground beef. Ph.D. Dissertation, Michigan State University, East Lansing, MI, USA Chen, Y., Zheng, R., Jia, Z., and In, Y. 1990. Flavonoids as superoxide scavengers and antioxidants. Free Radical Biol. Med. 9:19-21. Chen, 0, Pearson, A.M., and Gray, J .I. 1992. Effect of different antioxidants on formation of meat mutagens during frying of ground beef. Proc. 38th Intern. Congr. Meat Sci. Technol. 3:567—570, August 23-28, Clermont-Ferramd. France. Chen, B.H., and Meng, ON. 1999. Formation of heterocyclic amines in a model system during heating. J. Food Protect. 62: 1445-1450. Dashwood, R.H., Xu, M., Hemaez, J.F., Hasaniya, N., Youn, K., Razzuk, A 1999. Cancer chemopreventive mechanism of tea against heterocyclic anrine mutagens from cooked meat. Proc. Soc. Exp. Biol. Med. 220:239-243. De Flora, S., Benicelli, C., Serra, D., Izzotti, A, Cesarone, C. Role of glutathione and N-acetylcysteine as inhibitors of mutagenesis and carcinogenesis. In Absorption and utilization of arrrino acids, Friedman, M., Ed. CRC, Boca Raton, FL, 1989. 3: 19-53. Deshpande, R., Khan, M., Bhat, D., and Navalkar, R 1993. Inhibition of Mycobacterium avium complex isolates from AIDS patients by garlic (Allium sativum). J. Antinricrob. Chemother. 32:623 -626. Dragsted, L0. 1992. Exposure and carcinogenicity of heterocyclic amines. Proceedings of the Toxicology Forum, 1992 Annual Meeting, Copenhagen, Denmark, 141- 148. Edmonds, C.G., Sethi, S.K., Yamaizumi, Z., Kasai, H., Nishimura, S., and McCloskey, J.A. 1986. Analysis of mutagen from cooked foods by directly combined liquid chromatography-mass spectrometry. Environ. Health Perspect. 67 :35-40. Esumi, H., Ohgaki, H., Kohzen, E., Takayarna, S., and Sugimura, T. 1989. Inducflon of lyinphoma in CDFI mice by the food mutagen, 2-arnino-lmethyl-6-phenyl- imidazo[4,5—b]pyridine. Jpn. J. Cancer Res. 80:1176-1178. Faulkner, J .A. 1994. Mechanism of heterocyclic amine formation in flied ground beef- the role of oxidized lipid and the Maillard reaction. Ph.D. Dissertation. Michigan State University, East Lansing, MI USA. 117 .,_a......_...1 Felton, J.S., Knize, M.G., Wood, C., Wuebbles, B.J., Healy, S.K., Stuermer, D., Bjeldanes, L.F., Kimble, BI, and Hatch, RT. 1984. Isolation and characterization of new mutagens flom flied ground beef. Carcinogenesis 5:95- 102. Felton, J.S., Knize, M.G., Shen, N.H., Anderson, B.D., Bjeldanes, L.F., and Hatch, F .T. 1986a. Identification of the mutagens in cooked beef. Environ, Health Perspect. 67:17-24. Felton, J.S., Knize, M.G., Shen, N.H., Lewis, PR, Andresen, B.D., Happe, J., and Hatch, F .T. 1986b. The isolation and identification of a new mutagen flom firied ground beef. 2-amino-l-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). Carcinogenesis 7: 1081-1086. Felton, J .S., and Knize, MG. 1990. Heterocyclic amine mutagens/carcinogens in foods. In H_andbook of Experimental Pharmacology. Eds, C.S. Cooper and PL. Grover. pp.471-502. Springer-Veflag, Berlin. Felton, J .S., and Knize, MG. 1991. Occurrence, identification, and bacterial mutagenicity of heterocyclic amines in cooked food. Mutation Res. 259:205-217. Felton, J.S., Knize, M.G., Roper, M., Fultz, E., Shen, NH, and Turteltaub, K.W. 1992. Chemical analysis, prevention and low-level dosirnetry of heterocyclic amines flom cooked food. Cancer Res. (Suppl) 5222103 s-2107s. Felton, J.S., Fultz, E., Dolbeare, F .A., and Knize, MG. 1994. Effect of microwave pretreatment on heterocyclic aromatic amine mutagens/carcinogens in flied beef patties. Food Chem. Toxicol. 32:897-903. Felton, J.S., Malfatti, M., Knize, M.G. Salmon, C.P., Hopmans, E., and Wu, R. 1997.. Health risks of heterocyclic amines. Mutation Res. 376237-41. Folch, J., Lees, M., and Stanley, G. H. 1957. A simple method for the isolation and purification of total lipids flom animal tissues. J. Biol. Chem. 226:497-509. Friedman, M., Wehr, C., Schade, J ., MacGregor, J. 1982. Inactivation of aflatoxin B1 mutagenecity by thiols. Food Chem. Toxicol. 20:887-892. Friedman, M., and Molnar-Perl, I. 1990. Inhibition of browning by sulfur amino acids. 1. heated anrino acid-glucose system. J. Agric. Food Chem. 38: 1642-1647. Friedman, M. 1996. Food browning and its prevention: An overview. J. Agric. Food Chem. 44:631-653. 118 Ghannonoum, M. 1988. Studies on the anticandidal mode of action of Allium sativum (garlic). J. Gen. Microbiol. 134:2917—2924. Grivas, S., Nyharnmar, T., Olsson, K., and JCDgerstad, M. 1985. Formation of a new mutagenic DiMeIQx compound in a model system by heating creatinine, alanine and fructose. Mutation Res. 151:177-183. Grivas, S., Nyharnmar, T., Olsson, K., and J @gerstad, M. 1986. Isolation and identification of the food mutagens IQ and MeIQx flom a heated model system of creatinine, glycine, and glucose. Food Chem. 20:127-136. Gross, G.A, Philippossian, G., and Aeschecher, H.U. 1989. An eficient and convenient method for the purification of mutagenic heterocyclic amines in heated meat products. Carcinogenesis 10:1175-1182. Gross, GA 1990. Simple methods for quantifying mutagenic heterocyclic aromatic amines in food products. Carcinogenesis. 11:1597-1603. Gross, GA, and Griiter, A. 1992. Quantitation of mutagenic/carcinogenic heterocyclic amines in food products. J. Chromatogr. 592:271-278. Gross, G.A, Turesky, RJ., Fay, B.L., Stillwell, W.G., Skipper, PL, and Tarrnenbaum, SR 1993. Heterocyclic aromatic anrine formation in grilled bacon, beef and fish and in grill scrapings. Carcinogenesis 14:2313-2318. Gry, J., Vahl, M., and Nielsen, P. 1986. Mutagens in Fried Meat Publ. No. 139, National Food Administration, Copenhagen, Denmark. Hargraves, W.A., and Pariza, M.W. 1983. Purification and mass spectral characterization of bacterial mutagens flom commercial beef extract. Cancer Res. 43:1467-1472.- Hatch, E.T., Felton, J .S., and Bjeldanes, LP. 1982. Mutagens flom the cooking of food: Themric mutagens in beef. In Carcinogens and Mutagens in the Environment. Vol.1. Ed, Stich, HF. pp. 147-173. Critical Reviews of Toxicology. CRC Press, Boca Raton, FL. Hayatsu, H, Arimotao, S., and Wakabayashi, K. 1991. Methods for separation and detection of heterocyclic amine. In Mutgens in Food & Detection 'a_ngl_ Prevention. Eds, H. Hayatsu. pp. 101-112. CRC Press, Boca Raton, FL. Hirose, M., Takahashi, S., Ogawa, K., Futakuchi, M., and Shirai, T. 1999. Phenolics: Blocking agents for heterocyclic amine-induced carcinogens. Food Chem. Toxicol. 37:985-992. 119 ’ In Ho, C.T., Chen, Q., Shi, H., Zhang, K.Q., and Rosen, RT. 1992. Antioxidative effect of . polyphenol extract prepared fl'om various Chinese teas. Prev. Med. 21 :520-525. Hodge, J. E. 1953. Chemistry of browning reactions in model systems. J Agric. Food Chem. 1: 928- 943. Holme, J ., Walline, H., Brunborg, G., Soderlund, E., Hongslo, J ., and Alexander, J. 1989. Genotoxicity of the food mutagen 2-amino-1-methyl-6-phenylimidazo [4,5- b] pyridine (PhIP): formation of 2-hydroxyamino-PhIP, a directly acting genotoxic metabolite. Carcinogenesis. 10:1389-1395. Hughes, B., and Lawson, L. 1991. Antimicrobial effects of Allium sativum L. (garlic), Allium ampeloprasum (elephant garlic), and Allium cepa L. (onion), garlic compounds and commercial garlic supplement products. Phytother. Res. 5:154- 158. Iberl, B, kaler, G, and Knobloch, K. 1990. Products of allicin transformation: ajoenes and dithiins, characterization and their determination by HPLC. Planta. Med. 56: 202-211. lagerstad, M., Reutersward, L.A, Olsson, R., Grivas, S., Nyharnmer, T., Olsson, K., Oste, R., and Dahlqvist, A. 1983a. Creatin(in)e and Maillard reaction products as precursors of mutagenic compounds: efl‘ects of various amino acids. Food Chem 12:25 5-264. Jagerstad, M., Reutersward, L.A., Oste, R, Dahlqvist, A, Grivas, S., Olsson, K., and Nyhammar, T. 1983b. Creatinine and Maillard reaction products as precursors of mutagenic compounds formed in flied beef. In The Maillard Regction in Food gd Nutrition. ACS Symposium Series 215. Eds, Walleer, G., and Feather, M. pp.507-519. American Chemical Society, Washington, DC. Jackson, S., Hargraves, A, Stroup, W., and Diachenko, G. 1994. Heterocych aromatic amine content of selected beefflavors. Mutation Res. 320: 1 13-124. Johansson, M.A.E., and Jagerstad, M. 1993. Influence of oxidized deep-flying fat and iron on the formation of food mutagens in a model system. Food Chem. Toxicol. 31:971-979. Johansson, M.A.E., and lagerstad, M. 1994. Occurrence of mutagenic/carcinogenic heterocyclic amines in meat and fish products, including pan residues, prepared under domestic conditions. Carcinogenesis 15: 151 1-1518. Jones, RC, and Weisburger, J .H. 1988. Inhibition of aminoimidazoquinoxaline type and aminoimidazol-4-one—type mutagen formation in liquid reflux models by L- tryptophan and other selected indoles. Jpn. J. Cancer Res. 79:222-230. 120 ' Kabelik, J. 1970. Antimikrobielle eigenschafien des knoblauchs. Pharmazie. 25:266- 270. Kamanna, V, and Chandrasekhara, N. 1984 Hypocholesteremic activity of different ’ fractions of garhc Indian J. Med. Res. 79: 580-583. , Kasai, H., Yatnaizumi, Z., Wakabayashi, K., Nagao, M., Sugitnura, T., Yokoyama, S., Miyazawa, T., Spingam, N., Weisburger, J ., and Nishimura, S. 1980. Potent novel mutagens produced by broiling fish under normal conditions. Proceedings of the Japan Academy 56: 278-283. Kasai, H., Yamaizumi, Z., Nishimura, S., Wakabayashi, K., Nagao, M., Sugimura, T., spingam, N., Weisburger, J ., Yokoyama, S., and Miyazawa, T. 1981a. A potent mutagen in broiled fish. Part1. 2—amino-3 methyl-3H-imidazo(4,5-f)quinoline. J. Chem. Soc. 2290-2293. Kasai, H., Ywnaizumi, Z., Shiomi, T., Yokoyama, S., Miyazawa, T., Wakabayashi, K., Nagao, M., Sugimua, T., and Nishimura, S. 1981b. Structure of a potent mutagen isolated from fried beef. Chem. Lett. 4:485-488. Kato, T., Kikugawa, K, and Hayatsu, H. 1986. Occurrence of the mutagens 2-amino- 3,4,8-trimethyl-imidazo(4,5-f)quinoxaline (4,8-MeIQx) in Japanease smoked, dried fish products. J. Agric. Food Chem. 34:810-814. Kato, T., Michikoshi, K., Minowa, Y., Maeda, Y., and Kikugawa, K. 1998. Mutagenicity of cooked hamburger is reduced by addition of onion to ground beef. 'Mutation Res. 420:109—114. Kendler, B. 1987. Garlic (Allium sativium) and onion (Allium cepa): A review of their relationship to cardiovascular disease. Prev. Med. 16:670-685. Kikugawa, K., Kato, T, and Hayatsu, H 1986. The presence of 2-amino-3, 8- dimethylimidazo[4, 5 -f]quinoxaline in smoked dry bonito (katsuobshi). Jpn. J. Cancer Res. 7: 99-102. Kim, 18., Wakabayashi, K., Kurosaka, R, Yamaizumi, Z., Jinno, F., Koyota, S., Tada, A., Nukaya, H., Takahashi, M., Sugimura, T., and Nagao, M. 1994. Isolation and identification of a new mutagen, 2-amino—4-hydroxy-methyl—3,8-dimethylinridazo (4,5-j)quinoxaline (4-CH20H-8-MeIQx), from beef extract. Cacinogenesis. 15:21- 26. Kitada, M., Taneda, M., Ohta, K., Nagashima, K., Itahashi, K., and Kamataki, T. 1990. Metabolic activation of aflatoxin B1 and 2-an1ino-3-methyl-in1idazo[4,5- flquinoline by human adult and fetal livers. Cancer Res. 50:2641-2645. 121 Kitada, M., Taneda, M., Itahashi, K., and Karnataki, T. 1991. Forms of cytochrome P450 in human fetal liver: purification and their and their capacity to activate promutagens. Jpn. J. Cancer Res. 82:426—432. Knize, M.G., Roper, M., Shen, RN, and Felton, J.S. 1990. Proposed structure for an amino-dimethylimidazofirropyridine mutagen in cooked meats. Carcinogenesis. 1 1 :2259-2262. Knize, M.G., Hopmans, E., and Happe, J .A. 1991. The identification of a new heterocyclic amine mutagen from a heated mixture of creatine, glutamic acid and glucose. Mutation Res. 260:3 13-319. Knize, M.G., Dolbeare, F.A., Carroll, K.L., Moore, DH, and Felton, J.S. 1994. Effect of cooking time and temperature on the heterocyclic amine content of fried-beef patties. Food Chem. Toxicol. 32:595-603. Knize, M.G., Salmon, C.P., Hopmans, EC, and Felton, J .S. 1997. Analysis of foods for heterocyclic aromatic amine carcinogens by solid-phase extraction and high performance liquid chromatography. J. Chromatogr. 763: 179-185. Knize, M.G., Sinha, R, Brown, B.D., Salmon, C.P., Levander, 0A, Felton, J.S., and Rothman, N. 1998. Hetrocyclic amine content in restaurant-cooked hamburgers, steaks, ribs, and chicken. J. Agric. Food Chem. 46:4648-4651. Knize, M.G., Salmon, C.P., Pals, P., and Felton, J.S. 1999. Food heating and the formation of heterocyclic aromatic amine and polycyclic aromatic hydrocarbon mutagens/carcinogens. Adv. Exp. Med. Biol. 459: 179-193. Knowles, L., and Milner, J. 1998. Depressed p34cdc kinase activity and G2/M phase arrest induced by diallyl disulfide in HCT-15 cells. Nutrition and Cancer. 30: 169- 174. Kritchevsky. D. 1991. The effect of dietary garlic on the development of cardiovascular disease. Trends Food Sci. Technol. 2:141-144. Krone, C., and Iwaoka, W., 1987. Commercial food processing operation and mutagen formation. J. Food Protect. 50:167-173. Kurosaka, R, Wakabayashi, K., Ushiyama, H., Nukaya, H., Arakawa, N., Sugimura, T., and Nagao, M. 1992. Detection of 2-amino-l-methyl-6-(4-hydroxyphenyl)- imidazo[4,5-b]pyridine in broiled beef. Jap. J. Cancer Res. 83:919-922. Lansen, J., Dragsted, L., Frandsen, H., Kristiansen, E., Rasmussen, E. Nielsen, P., and Knudsen, I. 1990. Carcinogenicity of mutagen from cooked meats. In Mut_agen and Carcinogens in the Diet. Eds, Pariza, M.W., Aeschbacher, J., Felton, J.S., and , Sato, S. pp. 89-108. Wiley-Liss, New York, NY. 122 u] ” .. Lawson, L., and Hughes, B. 1990. Trans-l-propenyl thiosulfinates: new compounds in garlic homogenates. Planta. Med. 56:589-596. Lawson, L., Wood, S., and Hughes, B. 1991. HPLC analysis of allicin andd other thiosulfinates in garlic clove homogenates. Planta. Med. 57:263-270. Lawson, L., Ransom, D., and Hughes, B. 1992. Inhibition of whole blood platelet aggregation by compounds in garlic clove extracts and commercial garlic . products. Thromb. Res. 65:141-156. Lawson, L. 1993. Bioactive organosulfur compounds of garlic and garlic products; role in reducting blood lipids. In Human Medicinal Agents from Plants. ACS Symp_. Ser. 534. Eds, AD. Kinghom and MR Balandrin, pp. 306-330. Am. Chem. Soc. Books, Washington, DC. Lawson, L. 1996. The composition and chemistry of garlic cloves and processed garlic. Ch. 3 In G_ar1ic; The Science: and Therapeutic Appligartion of Allium sativum L. and Related Sm’es. Eds, H.P. Koch and LB. Lawson, pp. 37-69. Williams & Wilkins, Baltimore, Maryland. Lee, H., and Tsai, SJ. 1991. Detection of IQ-type mutagens in canned roasted eel. Food Chem. Toxicol. 29:517-529. Lucier, G., and Lin, B. 2000. Gariic: Flavor of the ages. Agric. Outlook Jurre-July, 7- 10. Lutomski, J. 1983. Das wichtigste uber knoblauch und knoblauch-praparate. Dtsch. Apoth. Ztg. 123:623-624 Lynch, AM, Knize, M.G., Boobis, AR, Gooderharn, N.J., Davies, D.S., and Murray, S. 1992. Interindividual variability in systemic exposure in humans to 2-amino- 3,8-dimethylirnidazo(4,5-f)qrrinoxaline and 2-arnino-1-methyl-6-phenylimidazo (4,5-b)pyridine, carcinogens present in cooked beef. Cancer Res. 52:6216-6223. Manabe, S., Kurihara, N., Wada, O., Tohyama, K., and Aramaki, T. 1992. Formation of PhIP in a mixture of creatine, phenylalanine and sugar or aldehyde by aqueous heating. Carcinogenesis 13:827-830. Milic, B.L., Djilas, S.M., and Canadanovic-Brunet, J.M. 1993. Synthesis of some heterocyclic amino-imidazoazarenes. Food Chem. 46:273-276. Milner, J. 1996. Garlic: its anticarcinogenic and antimutagenic properties. Nutr. Rev. 54:882-S86. 123 Mohammad, S. and Woodward, S. 1986. Characterization of a potent inhibitor of platelet aggregation and release reaction isolated from Allium safivum (garlic). Thromb. Res. 44:793-806. Moore, D., and Felton, J.S. 1983. A microcomputer program for analyzing Ames test data. Mutation Res. 119:95-102. Mottram, D.S., and Whitfield, EB. 1995. Maillard-lipid interactions in nonaqueous systems: Volatiles from the reaction of cysteine and ribose with phosphatidylcholine. J. Agric. Food Chem 43: 1302-1306. Muramatsu, M., and Matsushima, T. 1985. Formation of MeIQx and 4,8-DiMeIQx by heating mixtures of- creatinine, amino acids, and monosaccharides. Mutation. Res. (Abstract) 147:266-267. Murkovic, M., Steinberger, D., and Pfannhauser, W. 1998. Antioxidant spices reduce the formation of heterocyclic amines in flied meat. Z. Lebensm. Unters. Forsch. A 207 :477-480. Murray, 8., Gooderham, N.J., Boobis, AR, and Davies, D.S. 1987. Trp-P-2 is not detectable in cooked meat and fish. Carcinogenesis. 8:937-940. Murray, S., Gooderharn, N.J., Boobis, AR, and Davies, D.S. 1988. Measurement of Melox and DiMeIQx in fiied beef by capillary column gas chromatography electron capture negative ion chemical ionization mass spectrometry. Carcinogenesis 9:321-325. Murray, 8., Gooderham, N.J., Boobis, AR, and Davies, D.S. 1993. Quantification of the carcinogens 2-amino-3,8-dimethyl-and 3,4,8-trimethylimidazo(4,5- flquinoxaline and 2-amino-1-methylimidazo(4,5-b)pyridine in food using a combined assay based on capillary column gas chromatography-negative ion mass spectrometry. J. Chromatogr. 616:222-219. Namiki, M., and Hayashi, T. 1975. Development of novel fi'ee radicals during the amino-carbonyl reaction of sugars with amino acids. J. Agric. Food Chem. 23(3):487—493. Namiki, M., and Hayashi, T. 1980. Formation of two-carbon sugar fragment at an early stage of the browning reaction of sugar with amine. Agric. Biol. Chem. 44(11):2575-2579. Namiki, M., and Hayashi, T. 1981. Formation of novel free radical products in an early stage of Maillard reaction. Progr. Food Nutr. Sci. 5:81-91. Namiki, M., and Hayashi, T. 1983. A new mechanism of the Maillard reaction involving sugar fragmentation and free radical formation. In The Maillard 124 -' 2“” Pi; 5.11 u‘ Ii I . .. Reaction i_n Foods and Nutrition. Eds, G.R Waller and MS. Feather. pp.21-46. American Chemical Society, Washington, DC. Negishi, C., Wakabayashi, K., Tsuda, M., Sato, S., Sugimura, T., Saito, H., Maeda, M., and Jagerstad, M. 1984. Formation of 2-amino-3,7,8-trimethyl[4,S-flquinoxaline, a new mutagen, by heating mixture of creatine, glucose, and glycine. Mutation Res. 140:55-59. Neter, J ., Wasserman, W., and Whitmore, GA. 1993. Analysis of variance. In Applied Statistics. pp 651-683. Simon and Schuster, Inc., Nwdham Heights, MA. Nielson, K., Mahoney, A, Williams, L., and Rogers, V. 1991. X-Ray fluorescence measurements of Mg, P, S, Cl, K, Ca, Mn, Fe, Cu, and Zn in fi'uits, vegetables and grain products. J. Food Comp. Analysis. 4:39-51. Nishimura, H., Wijava, C. H., and Mizutani, J. 1988. Volatile flavor components and antithrombotic agents: Vinyldithiins from Allium victorialis. J. Agric. Food Chem. 36:563-566. Nukaya, H., Koyota, S., Jinno, F., Ishida, H., Wakabayashi, K., Kurosaka, R, Kim, I., Yamaizumi, Z., Ushiyama, H., Sugimura, T., Nagao, M., and Tsuji. 1994. Structural determination of a new mutagenic heterocyclic amine, 2-amino-1,7,9- trimethylimidazo[4,5-g]quinoxaline (7 ,9-DiMeIng), present in beef extract. Cacinogenesis. 15:1151-11'54. Nyhammar, T. 1986. Studies on the Maillard reaction and its role in the formafion of food mutagens. Doctoral Thesis, Swedish University of Agricultural Sciences, Uppsala, Sweden ISBN 91-576-2658-8. ‘ Oguri, A, Suda, M., Totsuka, Y., Sugimura, T., and Wakabayashi, K. 1998. Inhibitory efi‘ects of antioxidants on formation of heterocyclic amines. Mutation Res. 402:23 7-245. Ohgaki, H., Kusarn, K., Matsukura, N., Morino, K., Hasegawa, H., Sato, S., Sugimura, T., and Takayama, S., 1984. Carcinogenicity in mice of a mutagenic compound, 2- arnino-3,4—dimethylimidazo[4,5-f]quinoline, from broiled sardine, cooked beef and beefextract. Carcinogenesis. 5:921-924. Ohgaki, H., Hasegawa, H., Suenaga, M., Kato, T., Sato, S., Takayama, S., and Sugimura, T. 1986. Induction of hepatocellular carcinoma and highly metastatic. squamous cell carcinomas in the forestth of mice feeding 2-amino-3,4- dimethylimidazo[4,5-j]quinoline. Carcinogenesis. 5:921-924. Ohgaki, H., Hasegawa, H., Suenaga, M., Sato, S., Takayama, S., and Sugimura, T. 1987. Carcinogenicity in mice of a mutagenic compound (MeIQx) from cooked foods. Carcinogenesis. 8:665-668. 125 . —__"‘_I' I Ohgaki, H., Takayarna, S., and Sugimua, T. 1991. Carcinogenicities of heterocyclic amines in cooked food. Mutation Res. 259:399-410. Okarnoto, T., Shudo, K., Hashimoto, Y., Kosuge, T., Sugimura, T., and Nishimura, S. . 1981. identification of a reactive metabolite of the mutagen, 2-amino-3- methylimidazo(4,5-j)-quinoline. Chem. Pharm. Bull. 29:590-593. Omkumar, R, Kadam,‘ S., Banerji, A, and Ramasarma, T. 1993. On the involvement of intramolecular protein disulfide in the irreversible inactivation of 3-hydroxy-3- methylglutaryl-CoA reductase by diallyl disulfide. Biochimica et Biophysica Acta. 1164:108-112. Orth-Wagner, S. 1986. Moderne phytotherapie. Dtsch. Apotheker. 38:42-47. Overvik, E, Kleman, M., Berg, 1., and Gustafsson, J. 1989. Influence of creatine, amino acids and water on the forinafion of the mutagenic heterocyclic amines found in cooked meat. Carcinogenesis 10:2293-2301. Paterson, A.M., and Chipman, J .K. 1987. Activation of 2-amino-3-methylimidazo—(4,5- flquinoline in rat and human hepatocyte/ Salmonella mutagenicity assays: the contribution of hepatic conjugation. Mutagenesis. 2: 137-140. Pentz, R, Guo, Z., Kress, G., Mfiller, B., and Siegers, GP. 1990. Standardization of garlic powder preparations by the estimation of fiee and hydrolysable SH groups. Planta. Med. 56:591-598. Plengvidhay, C., Chinayon, S., Sitprija, S., Pasatrat, S., and Tankeyoon, M. 1988. Effects of spray dried garlic preparation on primary hyperlipoproteinemia. J. Med. Assoc. Thailand. 71:248-252. Powrie, W., Wu, C., Rosin, M., and Stich, H. 1981. Clastogenic and mutagenic activities of Maillard reaction model systems. J. Food Sci. 46: 1433-1438. Pratt, D.E., and Birac, RM. 1979. Source of antioxidant activity of soybeans and soy products. J. Food Sci. 44:1720-1722. Rappaport, S.M., McCartney, M.G., and Wei, ET. 1979. Volatilization of mutagens from beef during cooking. Cancer Lett. 8: 139-145. Reistad, R, Possland, O., Latva-Kala, K., Rasmussen, T., Vikse, R, Becher, G., and Alexander, J .. 1997. Heterocyclic aromatic amines in human urine following a fried meat meal. Food Chem. Toxicol. 35:945-955. 126 Reuter, H. and Sendl, A 1994. Allium sativum and Allium ursinum: chemistry, pharmacology, and medical applications. In Economic and Medicirgl Plant Research. pp. 54-113, Academic Press, New York,. Reutersward, AL., Skog, K., and Jagerstad, M. 1987. Mutagenicity of pan-flied bovine tissues in relation to their content of creatine, creatinine, monosaccharides and flee amino acids. Food Chem. Toxicol. 25:755-762. Reynolds, T. 1963. Chemistry of nonenzyrnic browning. I. The reaction between aldoses and amines. Adv. Food Res, 12: 1-52. Rhee, K.S., Donnelly, KC, and Ziprin, Y.A 1987. Reduction of mutagen formation in flied ground beef by glandless cottonseed flour. J. Food Protect. 50:753-755. Rizzi, G. 1994. The Maillard reaction in foods. In Maillard Reactions in Chennm Food. and Health, Eds, T.P. Labuza, G.A Reineccius, V.M. Monnier, J. O'Brein, and J.W. Baynes., pp.11-19. The Royal Society of Chemistry, Thomas Graham House, Science Park, Cambridge. Saito, K., Shinohara, A, Kamatald, T., and Kato, R 1985. Metabolic activation of mutagenic N-hydroxyarylamines by O-acetyltranferase in Samonella ryphimurium TA98. Arch. Biochem. Biophys. 239:286-295. Salmon, C.P., Knize, M.G., and Felton, J.S. 1997. Effect of marinating on heterocych amine carcinogen formation in grilled chicken. Food Chem. Toxicol. 35:433-441. Schuirmann, E., and Eichner, K. 1991. Formation of arninoirnidazoquinolines ans quinoxalines (IQ-compounds) during heating of meat products. In Stategies for Food Quality Cormol and Analfiical Methods in Europe. Eds. W. Baltes, T. Eklund, R. Fenwich, W. Pfannhauseer, A Ruiter and HP. Their. pp.739-744. B.Behr’s Berlag, Hamburg. Scranton, L. 1997 . Efl‘ect of lipids and iron on heterocyclic aromatic amine formation in aqueous model systems. MS. Thesis. Michigan State University, East Lansing, MI, USA Semmler, F. 1892. Uber das Atherische Ol des Knoblauchs (Allium sativium). Arch. Pharm. 230:434—443. . Shashikanth, K., Basappa, S., and Sreenivasamurthy, V. 1986. Effect of feeding raw and boiled garlic (A Ilium sativum L.) extracts on the growth, caecal microflora, and serum proteins of albino rats Nutr. Rep. Int. 33 :3 13-3 19. Sheen, L.Y., Wu, C.C., Lii, C.K., and Tsai, SJ. 1999. Metabolite of diallyl disulflde and diallyl sulfide/in primary rat hepatocytes. Food Chem. Toxicol. 37: 1 139-1 146. 127 Shibamoto, T., Nishimura, O., and Millara, S. 1981. Mutagenicity of products obtained fi'om a maltol-armnonia browning model system J. Agric. Food Chem. 29:643- 646. Shioya, M., Wakabayashi, K., Sato, S., Nagao, M., and Sugimura, T. 1987. Formation of a mutagen, 2-amino-l-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in cooked beef, by heating a mixture containing creatinine, phenylalanine and glucose. Mutation Res. 191:133-138. Sichel, G., Corsaro, C., Scalia, M., Di Bilo, AJ., and Bonomo, RP. 1991. In vitro scavenger activity of some flavonoids and melanins against 02'. Free Radical Biol. Med. 1121-8. Sinha, R, Knize, M.G., Salmon, C.P., Brown, B.D., Rhodes, D., Felton, J.S., Levander, O.A, and Rothman, N. 1998. Heterocyclic amine content of pork products cooked by difl'erent methods and to varying degree of doneness. Food Chem. Toxicol. 36:289-297. Singh, A, and Shukla, Y. 1998. Antitumor activity of diallyl sulfide on polycych aromatic hydrocarbon-induced mouse skin carcinogenesis. Cancer Letters. 131 :209-214. Sitprija, S., Plengvidhya, C., Kangkaya, V., Bhuvapanich, S., and Tunkayoon, M. 1987. Garlic and diabetes mellitus phase H clinical trial. J. Med. Assoc. Thailand. 70:223-227. Skog, K., and lagerstad, M. 1990. Efl‘ects of monosaccharides and disaccharides on the formation of food mutagens in model systems. Mutation Res. 25:263-272. Skog, K, and lagerstad, M. 1991. Effect of ghrcose on the formation of PhIP in a model system. Carcinogenesis. 12:2297-2300. Skog, K., J agerstad, M. and Laser-ReutersWard, A 1992. Inhibitory effect of carbohydrates on the formation of mutagens in flied beef patties. Food Chem. Toxicol. 30:681-688. Skog, K. 1993. Cooking procedures and food mutagens: A literature review. Food Chem. Toxicol. 31:655-675. Skog, K., and Jagerstad, M. 1993. Incoporation of carbon atoms flom glucose into the food mutagens MeIQx and 4, 8-DiMeIQx using MC—labelled glucose in a model system. Carcinogenesis. 14:2027-2031. Skog, K., Steineck, G., Augustsson, K., and lagerstad, M. 1995. Effect of cooking temperature on the formation of heterocych amines in flied meat products and pan residues. Carcinogenesis. 16:861-867. 128 Skog, K., Augustsson, K., Steineck, G., Stenberg, M., and Jagerstad, M. 1997. Polar and nonpolar heterocyclic amines in cooked fish and meat products and their corresponding pan residues. Food Chem. Toxicol. 35:555-565. Skog, K., Solyakov, A, Arvidsson, P., and lagerstad, M. 1998. Analysis of nonpolar heterocyclic amines in cooked foods and meats extracts using gas chromatography-mass spectrometry. J. Chromatogr. 803:227-233. Skog, K., Solyakov, A, and lagerstad, M. 2000. Efl‘ects of heating conditions and additives on the formation of heterocyclic amines with reference to amino- carbolines in a meat juice model system. Food Chem. 68:299-308. Smith, AR, and Circle, SJ. 1978. Soybeans. In QM’ and Technology. Avi Publishing Co., Westport, CT. Snyderwine, B.G., Roller, P.P., \Vrrth, P.J., Adarnson, R.H., Sato, S., and Thorgeirsson, SS. 1987. Synthesis, purification and mutagenicity of 2-hydroxyamino-3- methylimidazo[4,5-j]quinoline. Carcinogenesis 8: 1017-1020. Sorata, Y., Takahama, U., Kimura, M. 1984. Protective effect of quercetin and rutin on photosensitized lysis of human erythrocytes in the presence of hematoporphyrin. Biochem. Biophys. Acta. 799:313-317. Spamins, V.L., Barany, G., and Watternberg, L.W. 1988. Eifects of organosulfirr compounds from garlic and onions on benzo[a]pyreneinduced neoplasia and glutathione-S-transferase activity in the mouse. Carcinogenesis. 9: 131-134. Spingarn, N. E., and Garvie, C. T. 1979. Formation of mutagens in sugar-ammonia model systems. J. Agric. Food Chem. 27: 1319-1321. Stavric, B. 1994. Biological significance of trace levels of nmtagenic heterocych aromatic amines in human diet: a critical review. Food Chem. Toxicol. 32:977- 994. Stoll, A. and Seebeck, E. 1947. Uber Allin, die genuine Muttersubstanz des Knoblauchéls. Experientia 31114-1 15. Sugii, M., Suzuki, T. ,Nagasawa, S, and Kawashima, K 1964. Isolation of gamma- -L- glutarnyl- S-allylmercapto-L-cysteine and S-allylmercapto-L-cysteine from garlic. Chem. Pharm. Bull. 12: 1111- 1115. Suginnrra, T., and Sato, S. 1982. Mutagens, carcinogens, and tumor promoters in our daily food. Cancer. 49: 1970-1984. 129 I.‘ — i .. Sugimura, T., Sato, S., and Wakabayashi, K. 1988. Mutagens/carcinogens in pyrolysates of amino acids and proteins and in cooked foods: heterocyclic aromatic amines. In Chemical Induction of Cancer. Structufll Basis and Biological Mechanism. Eds, V. T. Woo, D. V. Lai, J.C. Arcos and M. F. Argus. pp.681-710. Academic Press, New York. Surh, Y.J., Lee, R'C., Park, K.K., Mayne, S.T., Liem, A, and Miller, J.A. 1995. Chemoprotective effects of capsaicin and diallyl sulfide against mutagenesis or turnorigenesis by vinyl carbamate and N-nitrosodirnethylamine. Carcinogenesis. 16:2467-2471. ' Suzuki, T., Sugii, M., and Kakimoto, T. 1961. New gamma-glutamyl peptides in garlic. Chem. Pharm. Bull. 9:77-78. Suzuki, T., Sugii, M., and Kakimoto, T. 1962. Garmna-Irghrtamyl-S-allyl-Ircysteine, a new gamma-glutamyl peptide in garlic. Chem. Pharm. Bull. 10:345-346. Tai, CY, and Ho, CT. 1998. Influence of glutathion oxidation on thermal formation of Maillard aromas. J. Agric. Food Chem. 46:2260-2265. Takahashi, M., Wakabayashi, K., Nagao, M., Yamamoto, M., Masui, T., Goto, T., Kinae, N., Tomita, I., and Sugimura, T. 1985. Quantification of 2-amino-3- methylimidazo[4,5-f]quinoline (IQ) and 2-amino-3-dimethylimidazo[4,5- flquinoxaline (MeIQx) in beef extracts by liquid chromatography with electrochemical detection (LCEC). Carcinogenesis 6:1195-1199. Takahashi, M., Toyota, K., Aze, Y., Furuta, K., Mitsumori, K., and Hayashi, Y. 1993. The rat urinary bladder as a new target of heterocyclic amine carcinogenicity: tumor induction by 3-amino-1-methyl-5H-pyrido[4,3,-b]indole acetate. Jap. J. Cancer Res. 84:852-858. Taylor, RT., Fultz, E., and Knize, MG. 1985. Mutagen formation in a model beef boiling system. 111. Purification and identification of three heterocyclic amine mutagens-carcinogens. J. Environ Sci. Health. 20:135-148. Terao, J ., Piskula, M., and Yao, Q. 1984. Protective effect of epicatechin, epicatechin gallate, and quercetin on lipid peroxidation in phospholipid bilayers. Arch. Biochem. Biophys. 308:278-284. Thiebaud, H.P., Knize, M.G., Kuzrnicky, P.A, Felton, J.S., and Hsieh, DP. 1994. Mutagenicity and chemical analysis of fumes flom cooking meat. J. Agric. Food Chem. 42:1502-1510. Thomson, B. 1999. Heterocyclic amine level in cooked meat and the implication for New Zealanders Euro. J. Cancer. Prev. 82201-206. 130 Troll, W., Frenkel, K., Mesner, R Protease inhibitors: their role as modifiers of the carcinogenic process. Adv. Exp. Med. Biol. 1986. 199:153-165. Trompeta, V., and O’Brien, J. 1998. Inhibition of mutagen formation by organosulfirr compounds. J. Agric. Food Chem. 46:4318-4323. Tsai, S.J., Jenq, SN, and Lee, H. 1996. Naturally occurring diallyl disulflde inhibits the formation of carcinogenic heterocyclic aromatic amines in boiled pork juice. Mutagenesis. 11:23 5-240. Turesky, RJ., “fishnok, J.S., Tannenbaum, S.R, Pfund, RA, and Buchi, G.H. 1983. Qualitative and quantitative characterization of mutagens in commercial beef extract. Carcinogenesis. 42863-866. Turesky, RJ., Bur, H., Huynh-Ba, T., Aeschbacher, H.U., and Milon, H. 1988. Analysis of mutagenic heterocyclic amines in cooked beef products by high- perforrnance liquid chromatography in combination with mass spectrometry. Food Chem. Toxicol. 26:501-509. Turesky, RJ., Forter, C.M., Aeschbacher, H.U., Wurzner, H.P., Skipper, P.L., Trudel, L.J., and Tannenbaum, SR 1989. Purification of the food-born carcinogens 2- amino-3-methylimidazo-(4,5-f)quinoline and 2-amino-3,8-dirnethylimidazo(4,5- flquinoxaline in heated meat products by immunoaflinity chromatography. Carcinogenesis 10:151-156. Ueda, Y., Kawajiri, H., Miyarnura, N., and Miyajima, R. 1991. Content of some sulfur- containing components and flee amino acids in various strains of garlic. J. Jpn. Soc. Food Sci. Technol. 38:429-434. Vahl, M., Gry, J., and Nielsen, P. A 1988. Mutagens in flied pork and the influence of flying temperature. (Abstract). Mutation Res. 203 :239. Virtanen, A, Hatanaka, M., and Berlin, M. 1992. Gamma-L-glutamyl-S-n- propylcystein in knoblauch (Allium sativum). Suom. Kenristil. B. 35:52-58. Virtanen, A 1965. Studies on organic sulfur compounds and other labile substances in plants. Phytochemistry. 4:207-228. Virtanen, A. 1969. Antimikrobielle und antithyreoide Stofi‘e in einigen Nahrungspflanzen. Qual. Plant. Mater. Veg. 1828-28. Wakabayashi, K., Takahasi, M., Nagao, M., Sato, S., Kinae, N., Tomita, 1., and Sugimura, T. 1986. Quantification of mutagenic and carcinogenic heterocych amines in cooked foods. Develop. Food Sci. 363-371. 131 Wakabayashi, K., Kim, I., Kurosaka, R, Yamaizumi, Z., Ushiyama, H., Takahashi, M., Koyota, S., Tada, A, Nukaya, H., Goto, S., Sugimura, T., and Nagao, M. 1995. Identification ‘of new mutagenic heterocyclic amine. In Heterocyclic Amine in Cooked Foods: Possible Hum_an (Larcinogens Eds, Adamsom, R, Gustavsson, J ., Ito, N., Nagao, M., Sugimura, T., Wakabayashi, K., and yamazoe, Y. pp. 197-206. Princeton Scientific Publishing Co., Princeton, NJ. Wakabayashi, K. and Sugimura, T. 1998. Heterocyclic amines formed in the diet: carcinogenicity and its modulation by dietary factors. J. Nutr. Biochem. 9:604- 612. Wang, Y.Y., Vuolo, L.L., Spingarn, NE, and Weisburger, J.H. 1982. Formation of mutagens in cooked foods: The mutagen reducing effect of soy protein concentrates and antioxidants during flying of beef. Cancer Lett. 16: 179-189. Wattenberg, L.W. Spamins, V.L., and Barany, G. 1989. Inhibition of N- nitrosodiethylamine carcinogenesis in mice by naturally occurring organosulfirr compounds and monoterpenes. Cancer Res. 49:2689-2692. Wedzicha, B. 1992. Chemistry of sulphiting agents in food. Food Addit. Cont. 9:449- 459. Wei, C.I., Kitamura, K., and Shibamoto, T. 1981. Mutagenicity of Maillard browning ‘ products obtained flom a starch-glycine model system. Food Cosmet. Toxicol. 19:749-751. Weisburger, J.H. 1991. Carcinogens in foods and cancer prevention. Adv. Exp. Med. Biol. 289:137-151. Weisburger, J.H., Nagao, M., Wakabayashi, K, and Oguri, A 1994. Prevention of heterocych amine formation by tea and tea polyphenols. Cancer Letters. 83 : 143- 147. Weiss, RF. 1986. Neues vom knoblauch. Arztez. Naturheilverf. 27:206-209. Wertheim, T. 1844. Untersuchung des Knoblauchols. Ann. Chem. Pharm. 51:289-315. Yamaizumi, Z., Shiomi, T., Kasai, H., Nisimura, S., Takahashi, Y., Nagao, M., and Sugimura, T. 1980. Detection of potent mutagens, Trp-P-l and Trp-P-2, in broiled fish. Cancer Lett. 9:7 5-83. Yamaizumi, Z., Kasai, H., Nishimura, S., Edmonds, C. G., and McCloskey, J. A. 1986. Stable isotope dilution quantification of mutagens in cooked foods by combined liquid chromatography-thermospray mass spectrometry. Mutation Res. 173 : 1-7. 132 37*“?! Yen, GO, and Chen, H.Y. 1995. Antioxidant activity of various tea extracts in relation to their antimutagenicity. J. Agric. Food Chem. 43:27-32. Yoshida, D., and Okarnoto, H. 1980. Formation of mutagens by heating creatine or amino acids with addition of fatty acids. Agric. Biol. Chem. 44:3025-3027. Yoshida, D., Sato, Y., and Mizusake, S. 1984. Isolation of 2-amino-3-methyl- imidazo[4,5-flquinoline as a mutagen flom the heated product of a mixture of creatine and proline. Agric. Biol. Chem. 48:241-847. Yoshida, S., Kasuga, S., Hayashi, N., Ushiroguchi, T., Matsuura, H., and Nakagawa, S. 1987. Antifungal activity of ajoene derived flom garlic. Appl. Environ. Microbiol. 53:615-617. Yu, T.H., Wu, C.W., and Ho, CT. 1994. Meat-like flavor generated flom thermal interaction of glucose and alliin or deoxyalliin. J. Agric. Food Chem. 42: 1005-1009. Zhang, X.M., Wakabayashi, K., Liu, Z.C., Sugimura, T., and Nagao, M. 1988. Mutagenic and carcinogenic heterocyclic amines in Chinese cooked foods Mutation Res. 201:181-188. 133 APPENDIX 1 3 E .1111: 08 030382 a». 833:3. v3.9.8.” on rough «388 a. Son m2 mo 53 134 real 1 OH 50. 1 . 1 0‘ i1 Mi 1 10 20 so 40 60 so 70 so so WW 1" 100 110 120 Mass spectrum of 5—(hydroxymethyl) 2-furancarboxaldehyde (Peak 1 on chromatogram). 135 100. 0 14 10 Mass spectrum of 2-furancarboxaldehyde (Peak 2 on chromatogram). 136 1004 3‘ Mass spectrum of 1,3-dihydroxy 2-propanone (Peak 3 on chromatogram). 137 roo. . V '5 Mass spectrum of Methyl 2-furoate (Peak 4 on chromatogram). 138 2. .3 .2 .3 _ e. , a: 93383.: on 883.5% 2.028.. 8 .596 eéigno a Son m2. 8 By a: 139 1m. 41 .50. J 1' _ ‘ , .. " rs o. h :1: . 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MH-Vhyl-tzdlrW-Gm Mass spectrum of 3-viny1-l ,2-dithiocyclohex-5-ene (Peak 3 on chromatogram). 142 INA OJ 1 1. 1 so so soon mama-2M ‘ I 3/\/s\5/s\/\ 1 . 17 70 so so 100 110 1201311140150 160 170 1110 Mass spectrum of diallyltrisulflde (Peak 4 on chromatogram). 143 Mass spectrum of 3-(2,3-dithia-5-hexenyl)-3,4-dihydro-2H-thiopyran (Peak 5 on chromatogram). 144 Mass spectrum of 6-methyl-4,5,8,9—tetrathiadodeca-1,1l-diene (Peak 6 on chromatogram). ,/\/ 100. 130.1 it 1 “2%. mlz 145 U I l I 2 so. rrfivr 300. 100. 80" Q/S‘S’M 60,- 4o.- 20.4 J 1 0.4 I ? ~ * ~ 0. 50. 100. . 1 u. 200. 21. 300. mlz ' Mass spectrum of 3-(2,3,4-trithia-6-heptenyl)—3,4-dihydro-2H-thiopyran (Peak 7 on chromatogram). 146 1] qfii Egéggégsggggéz 583.85! 3. E . ... Aw auvan. 147 100. ED * C7 I 504 45 ' ' ‘TI I W amt.-. ..... has“! ..... mil? 0 10 20 so (MW. I'm 50 so 70 N I) Mass spectrum of tetrahydrothiophene (Peak 1 on chromatogram). 148 501 1:1 7 | 11 . E 16 . 7. v' o. 1.; iii :4; 91 l ‘1 . 1‘ , .. 40 50 60 70 no so 100 110 120 1!) 140 150 160 170 1”. M1 {BA-Tm. M Mass spectrum of 3,5-diethyl-l,2,4-trithiolane (Peak 2 on chromatogram). 149 1WJ Tim 11H 41 ; "I111 ".1 :_x: 17 H 11. 6060100090 MW add. 31W)- 11.. 100 110 19 120 1:10 140 Mass spectrum of 3-(allylthio)-propionic acid (Peak 3 on chromatogram). 150 1W1 I vvvvvvvvv ' IIIIIIIII 'l 10 20 ~ 30 Wat-11W :l ' 74 102 100 Mass spectrum of tetrahydrothiophene-3 -one (Peak 4 on chromatogram). 151 100. 1a: 0'17TT53HT119PE. .1. VJ; Mass spectrum of 5-methyl-2-thiophene carboxaldehyde (Peak 5 on chromatogram). 152 10111 11. 1 12 -« c .4, ' I I 44 ‘WM 30 :0 60 N 70 N no 100110120 130140: -MO-W Mass spectrum of 9-thianoradamantane (Peak 6 on chromatogram). 153