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UBC Theses and Dissertations

Effect of diet modification on human fecal mutagenic activity Bell, Penelope Anne 1982

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EFFECT OF DIET MODIFICATION ON HUMAN FECAL MUTAGENIC ACTIVITY by PENELOPE ANNE BELL B.Sc. ( F . S c ) , M c G i l l U n i v e r s i t y , 1972 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES D i v i s i o n of Human N u t r i t i o n School of Home Economics We accept t h i s t h e s i s as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA J u l y , 1982 (£ ) Penelope Anne B e l l , 1982 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r reference and study. I f u r t h e r agree t h a t permission f o r extensive copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the head of my department or by h i s or her r e p r e s e n t a t i v e s . I t i s understood that copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l gain s h a l l not be allowed without my w r i t t e n permission. Department of Humg^ Aju-K- j 4- j ; e ^ The U n i v e r s i t y of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 DE-6 (3/81) ABSTRACT Dietary f a c t o r s have been implicated i n the e t i o l o g y of colon cancer. The s a l i e n t components of h i g h - r i s k d i e t s are thought to be high intakes of meat, e s p e c i a l l y beef, and f a t , e s p e c i a l l y animal f a t , and low intakes of f i b e r . Low-risk d i e t s are thought to be high i n f i b e r , and low i n meat and animal f a t . The present study examines the e f f e c t s of short-term consumption of d i e t s hypothesized to increase or decrease the r i s k f o r colon cancer on mutagenic a c t i v i t y of feces. Whether the f e c a l mutagens responsible f o r the mutagenic a c t i v i t y observed i n the study are d i r e c t l y involved i n the e t i o l o g y of colon cancer i s not known. However, most known mutagens are p o t e n t i a l l y carcinogenic, and f e c a l mutagenic a c t i v i t y may be an i n d i c a t o r of r i s k f o r colon cancer. Six healthy adult subjects consumed the following d i e t s i n sequence a baseline d i e t f o r one week, a low-risk lacto-ovo vegetarian, high f i b e r d i e t f o r two weeks, and a hig h - r i s k , high meat, low f i b e r d i e t f o r two weeks. Q u a n t i t a t i v e d a i l y food intake records were kept, and d a i l y bowel habits were recorded. Fecal samples were c o l l e c t e d at the end of each d i e t p e r i o d . Analyses were performed of the d i e t s f o r food and n u t r i e n t i n t a k e , and of feces f o r percent dry weight and pH. Mutagenic a c t i v i t y of the f e c a l samples was assayed using the f l u c t u a t i o n t e s t f o r mutagens. The subjects' h a b i t u a l d i e t s , although omnivorous, were found to c l o s e l y resemble a low-risk d i e t pattern. A n a l y s i s of the vegetarian and high meat d i e t s confirmed that the subjects had consumed foods which r e s p e c t i v e l y represented the components of h i g h - r i s k and low-risk d i e t s . The o v e r a l l f e c a l mutagenic a c t i v i t y obtained with samples on the high meat diet was higher than with the vegetarian or baseline diets using SaZmonolZa typhlmuJviium TA 98 and TA 100. The trend towards higher mutagenicity on the high meat diet over the vegetarian diet was consistent for a l l six subjects using TA 100, and for five of the six using TA 98. The vegetarian and baseline diets resulted in similar overall mutagenic activity. Analysis of the fecal sample parameters using the Kruskal-Wallis one-way analysis of variance showed no significant differences among fecal samples from the three diet periods with respect to wet weight, dry weight, percent dry weight, pH or number of daily bowel movements. However, a sign-test analysis showed a significant trend (p<0.05) towards fewer bowel movements on the high meat diet than on the vege-tarian diet. There were significant differences among subjects for a l l of the fecal sample parameters (p<0.01 or p<0.001). Spearman rank correlations were significantly positive between mutagenic acti v i t i e s using bacterial strains ScUbnone.ZZa ZyphAjmuJvlwm TA 98 and TA 100 for the baseline diet (p<0.01) and the vegetarian diet (p<0.05). There were also significant positive correlations (p<0.001) between pH and fecal mutagenicity on the high meat' diet using tester strain TA 100, and between wet weight and dry weight. The results of this study indicate that the overall mutagenic activity of human feces can be increased over a period of two weeks by the consumption of a diet high in meat and low in fiber, which i s considered to be a high-risk diet for colon cancer. i v TABLE OF CONTENTS Abstract i i L i s t of Tables v i i i L i s t of Figures x i Acknowledgements x i i Chapter I. Introduction 1 I I . Review of the L i t e r a t u r e 4 Organization of the Chapter 4 Mechanism to Identify Factors Involved i n Colon Carcinogenesis 4 Mutagenic Properties of Carcinogens ... 4 Sig n i f i c a n c e of Fecal Mutagens i n Colon Carcinogenesis 5 Role of Dietary Fat i n Colon Carcinogenesis 7 Dietary Fat Theory 7 Human Epidemiological Studies 9 Animal Studies 10 Evidence Against the Role of Dietary Fat i n Colon Carcinogenesis 11 Role of I n t e s t i n a l M i c r o f l o r a i n Colon Carcinogenesis 12 Human Epidemiological Studies 12 E f f e c t s of Diet on I n t e s t i n a l M i c r o f l o r a 13 Enzymatic A c t i v i t y of the I n t e s t i n a l M i c r o f l o r a 14 Role of Dietary Components i n Colon Carcinogenesis 16 E f f e c t s of Meat and Beef on Colon Carcinogenesis 16 E f f e c t s of Dietary Fat from Non-meat Sources on Colon Carcinogenesis 18 E f f e c t s of Vegetables on Colon Carcinogenesis 18 V Role of Dietary Fiber i n Colon Carcinogenesis 18 Fiber Theory 18 E f f e c t s of Fiber on Bowel Transit Time 20 E f f e c t s of Fiber on Fecal Bulk 22 E f f e c t s of Fiber Type on Fecal B i l e Acids 23 Evidence Against the Fiber Theory . 23 Chemical Nature of Fecal Mutagens, and I n h i b i t o r s 24 Possible Role for Nitroso-type Carcinogens 24 I n h i b i t o r s of Mutagenesis 27 Tests f o r Fecal Mutagens 27 Summary 29 I I I . Methods 31 Research Purpose and Plan 31 L i f e s t y l e Questionnaire and Dietary Data C o l l e c t i o n Instruments 32 L i f e s t y l e Questionnaire 32 Food Frequency Questionnaire 32 D a i l y Record Forms , 33 Description of Subjects 33 Information and Instructions Provided to Subjects 34 Dietary Guidelines and Information 34 Instructions for C o l l e c t i o n of Fecal Samples 34 Development of the Experimental Diets 35 Description of the Experimental Diets 35 Baseline Diet , , 35 Lacto-ovo Vegetarian Diet , , .. , 36 High Meat Diet , , 36 v i C o l l e c t i o n of Dietary Data and Fecal Samples 36 C o l l e c t i o n of Dietary Data 36 C o l l e c t i o n of Fecal Samples 37 Assays for Mutagenic A c t i v i t y of Feces 37 Preliminary Assays f o r Mutagenic A c t i v i t y 38 Fluctuation Test 38 Media and solutions 38 Bacteria 39 Preparation of f e c a l extracts 39 Test procedure 39 Determinations of Fecal Sample C h a r a c t e r i s t i c s 40 Dietary Analysis. 41 Analysis of Fecal Mutagenicity 42 S t a t i s t i c a l Analyses 42 S t a t i s t i c a l Analysis of Fecal Mutagenicity 43 Analysis of Parameters of Fecal Samples 43 Correlations Among Fecal Mutagenicity and Other Fecal Parameters 44 IV. Results and Discussion 45 Analysis of Diets 45 Analysis of Food Frequency Questionnaires 45 Comparison of Habitual and Baseline Diets , 48 Compliance with Guidelines for Experimental Diets 49 Composition of Baseline, Vegetarian and High Meat Diets 56 Analysis of Fecal Samples 65 Analysis of Mutagenic A c t i v i t y of Fecal Samples 65 Analysis of Fecal Sample C h a r a c t e r i s t i c s 77 v i i C orrelations Among Fecal Parameters 85 Correlations Among Fecal C h a r a c t e r i s t i c s 85 Correlations of Fecal Sample C h a r a c t e r i s t i c s with Mutagenicity 85 Correlations between Mutagenicity with Tester Strains TA 98 and TA 100 85 Summary ; 87 V. Summary, Limitations and Implications 88 Summary of the Study 88 Limitations of the Study 91 Implications 91 Bibliography 94 Appendices A. L i f e s t y l e Questionnaire 104 B. Food Frequency Questionnaire I l l C. Dai l y Food Record Form 119 D. Consent Form f or P a r t i c i p a t i o n i n the Study 121 E. Individual Dietary Guidelines 123 F. General Dietary Guidelines , 125 G. Diet Study Schedule Form 127 H. Instructions f o r C o l l e c t i o n of Fecal Samples 129 I. Background Studies Using Urine, Serum and Feces to In h i b i t Mutagenicity of Known Carcinogens 131 J. Results of Experiments to E s t a b l i s h S t a b i l i t y of Fecal Mutagens and Rep r o d u c i b i l i t y of Fluctuation Test Results 135 K. Individual Food Intakes of Subjects 1 to 6 on Baseline, Vegetarian and High Meat Diets . 137 L. Fluctuation Test Results f o r Individual Fecal Samples 156 v i i i LIST OF TABLES 1. Mean Daily Nutrient Intakes Derived from Food Frequency Questionnaires and Baseline Diets 46 2. Expected and Actual Mean Intakes of Selected Foods and Nutrients, Subject Number 1 50 3. Expected and Actual Mean Intakes of Selected Foods and Nutrients, Subject Number 2 51 4. Expected and Actual Mean Intakes of Selected Foods and Nutrients, Subject Number 3 52 5. Expected and Actual Mean Intakes of Selected Foods and Nutrients, Subject Number 4 53 6. Expected and Actual Mean Intakes of Selected Foods and Nutrients, Subject Number 5 54 7. Expected and Actual Mean Intakes of Selected Foods and Nutrients, Subject Number 6 55 8. Mean Dai l y Number and Range of Food Servings i n Baseline, Vegetarian and High Meat Diets f o r A l l Subjects 57 9. Mean D a i l y Nutrients i n Baseline, Vegetarian and High Meat Diets, Subject Number 1 58 10. Mean Daily Nutrients i n Baseline, Vegetarian and High Meat Diets, Subject Number 2 59 11. Mean Dai l y Nutrients i n Baseline, Vegetarian and High Meat Diets, Subject Number 3 60 12. Mean Daily Nutrients i n Baseline, Vegetarian and High Meat Diets, Subject Number 4 61 13. Mean Daily Nutrients i n Baseline, Vegetarian and High Meat Diets, Subject Number 5 62 14. Mean Daily Nutrients i n Baseline, Vegetarian and High Meat Diets, Subject Number 6 63 15. Mean and Range of Dai l y Nutrient Intakes on Baseline, Vegetarian and High Meat Diets f o r A l l Subjects 64 16. Composition of Diets Expressed as Percent of K i l o c a l o r i e s f o r Each Subject and the Group 66 17. Sum of Tubes Containing Revertant Bacteria f o r Each Diet Period at Each Extract Concentration 67 18. Average Number of Mutation Events per Fluctuation Tube on Each Diet at Each Extract Concentration 69 i x 19. Integrated Values for Mutation Events per Fluctuation Tube Expressed as a Dose-response Curve 71 20. Results of Chi-square Analyses at D i f f e r e n t Levels of Extract ... 72 21. Si g n i f i c a n c e of Chi-square Analyses at D i f f e r e n t Levels of Extract 73 22. Analysis of the Diet-induced Changes i n Mutagenicity at Each Extract Concentration and for Each Subject by the Sign-test for Matched Pairs 74 23. Average Sum of Revertant Tubes f o r Each Subject and Diet Period 76 24. Wet Weights i n Grams of 24-hour Fecal C o l l e c t i o n s 78 25. Dry Weights i n Grams and Percent Dry Weights of 24-hour Fecal C o l l e c t i o n s 79 26. Kruskal-Wallis One-Way Analysis of Variance by Ranks of Fecal Sample Variables 80 27. pH of 24-hour Fecal C o l l e c t i o n s 82 28. pH Measurement Taken of a Standard Buffer Solution, pH 7.00, Used as a Control for Electrode D r i f t 83 29. Spearman Rank Correlations for Fecal Sample Variables 86 1.1. Number of Revertant Bact e r i a per 10 7 Survivors 132 J . l . Number of Fluctuation Test Tubes Containing Revertant Bacteria i n Repeated Fluctuation Tests on One Sample of Fecal Extract, Concentration 2.08 yl/ml 136 K . l . Mean Daily Food Intakes by Number of Servings on Baseline Diet, Subject Number 1 138 K.2. Mean Daily Food Intakes by Number of Servings on Baseline Diet, Subject Number 2 139 K.3. Mean Daily Food Intakes by Number of Servings on Baseline Diet, Subject Number 3 140 K.4. Mean Daily Food Intakes by Number of Servings on Baseline Diet, Subject Number 4 141 K.5. Mean Dai l y Food Intakes by Number of Servings on Baseline Diet, Subject Number 5 142 K.6. Mean Daily Food Intakes by Number of Servings on Baseline Diet, Subject Number 6 , 143 K.7. Mean Dai l y Food Intakes by Number of Servings on Vegetarian Diet, Subject Number 1 144 K.8. Mean Dai l y Food Intakes by Number of Servings on Vegetarian Diet, Subject Number 2 145 K.9. Mean Daily Food Intakes by Number of Servings on Vegetarian Diet, Subject Number 3 146 K.10. Mean Daily Food Intakes by Number of Servings on Vegetarian Diet, Subject Number 4 147 K..11. Mean Daily Food Intakes by Number of Servings on Vegetarian Diet, Subject Number 5 148 K.12. Mean Dai l y Food Intakes by Number of Servings on Vegetarian Diet, Subject Number 6 149 K.13. Mean Dai l y Food Intakes by Number of Servings on High Meat Diet, Subject Number 1 150 K.14. Mean Daily Food Intakes by Number of Servings on High Meat Diet, Subject Number 2 151 K.15. Mean Daily Food Intakes by Number of Servings on High Meat Diet, Subject Number 3 152 K.16. Mean Dai l y Food Intakes by Number of Servings on High Meat Diet, Subject Number 4 153 K.17. Mean Dai l y Food Intakes by Number of Servings on High Meat Diet, Subject Number 5 154 K.18. Mean Daily Food Intakes by Number of Servings on High Meat Diet, Subject Number 6 155 L . l . Number of Fluctuation Test Tubes Containing Revertant Bacteria After 5 Days, Subject Number 1 157 L.2. Number of Fluctuation Test Tubes Containing Revertant Bacteria After 5 Days, Subject Number 2 158 L.3. Number of Fluctuation Test Tubes Containing Revertant Bacteria A f t e r 5 Days, Subject Number 3 159 L.4. Number of Fluctuation Test Tubes Containing Revertant Bacteria A f t e r 5 Days, Subject Number 4 160 L.5 Number of Fluctuation Test Tubes Containing Revertant Bacteria A f t e r 5 Days, Subject Number 5 161 L.6. Number of Fluctuation Test Tubes Containing Revertant Bacteria A f t e r 5 Days, Subject Number 6 162 x i LIST OF FIGURES 1. Conversion of Deoxycholic Acid to 20-methyl-cholanthrene 8 2. Postulated Relationship Between Dietary Fiber and Colon Cancer 21 3. The Nitroso Exchange Reaction 25 4. Dose-response Curves f or Mutation Events per Fluctuation Test Tube at Each Concentration of Extract on Baseline, Vegetarian and High Meat Diets 70 5. Mean Number of Dai l y Bowel Movements on Baseline, Vegetarian and High Meat Diets 84 ACKNOWLEDGEMENT S I s i n c e r e l y appreciate the contributions of a l l thO:se who made possible the completion of t h i s project: Dr. Harriet Kuhnlein, my advisor, who provided the idea f o r the project and the support and guidance needed throughout i t s development; Dr. Urs Kuhnlein, committee member, who p a t i e n t l y endured my 'growing pains' i n the laboratory and guided the data an a l y s i s ; Dr. Nancy Schwartz, committee member, who provided valuable advice i n the preparation of the manuscript; Frank Flynn, who as s i s t e d i n the computer ana l y s i s ; Beth Howarth and Bev Brown, who typed the t h e s i s ; P a t r i c i a Parsons, who prepared the fi g u r e s ; Marcia Hawes, who r e l i e v e d me of family r e s p o n s i b i l i t i e s i n desperate moments; Jon, my husband, whose a t t i t u d e throughout kept t h i s project i n perspective Vanessa and Brannen, who were too young to r e a l i z e the existence of ' l i f e a f t e r t h e s i s ' ; and f i n a l l y , the subjects, whose p a r t i c i p a t i o n made the study possible. CHAPTER I INTRODUCTION While the precise etiology of colon cancer i s incompletely understood, considerable evidence e x i s t s to implicate d i e t as a causative f a c t o r . Epidemiological studies do not support genetic p r e d i s p o s i t i o n or environ-mental contaminants as causative factors for colon cancer. Genetic t r a i t s cannot account for the low incidence of colon cancer i n developing s o c i e t i e s reported by Wynder (19.76).. Further, migrant populations assume the r i s k f o r colon cancer of t h e i r new place of residence, e s p e c i a l l y for second and subsequent generation migrants. Wynder et a l . (.1969) suggested that d ietary differences are l i k e l y to be responsible for the infrequency of colon cancer i n Japan, i t s increase i n Japanese immigrants to the United States, and the high rate among Americans. The presence of environ-mental contaminants does not explain why population subgroups i n the same geographical area have d i f f e r e n t incidences of colon cancer. Seventh-Day-Adventists compared with non- Seventh-Day-Adventists i n New York are examples of such groups (Reddy et a l . , 19.80a). The vegetarian d i e t s con-sumed by the Seventh-Day-Adventists are thought to be the factor responsible for the low rate of colon cancer i n t h i s group. The observation has been made that populations at h i g h - r i s k for colon cancer consume d i e t s high i n animal protein and f a t , and low i n f i b e r . Conversely, low-risk populations consume, diets low i n animal protein and f a t , but high i n vegetable protein and f i b e r (Wynder, 1975)_. Most recently, Rozen et a l . (1981). compared high-incidence and low-incidence I s r a e l i populations with the same genetic backgrounds, and found that the low-incidence population consumed a d i e t r i c h e r i n f i b e r , f r u i t s and vegetables. Armstrong and D o l l (1975) showed that the environmental factors most highly correlated with colon cancer rates, are dietary meat and animal protein. 2 To t a l f a t , meat and animal protein i n the d i e t s of population groups are highly correlated with one another. Thus, di e t appears to be a major environmental factor i n the etio l o g y of colon cancer. The existence of several stages i n colon carcinogenesis has been hypothesized. The stages were described by Zaridze (1981) as the develop-ment of an adenoma, the growth of the adenoma, and the appearance of malignancy i n the adenoma. H i l l et a l . (1978) postulated that d i e t , i n p a r t i c u l a r f a t and meat, has i t s e f f e c t at the second stage or growth of the adenoma, and i s therefore the promoting factor i n a m u l t i f a c t o r i a l etiology. Other factors may influence the other two stages. E l u c i d a t i o n of the chain of events which leads to the development of colon cancer implies the opportunity of intervention to prevent the mani-f e s t a t i o n of the disease. Indeed, the preventive p o t e n t i a l f o r cancers associated with environmental factors was estimated by Wynder (1976) to be as high as 90%. Mutagenic a c t i v i t y of feces i s thought to be an ind i c a t o r of r i s k f o r colon cancer, and studies by Land and Bruce (1978), Reddy et a l . (1980b) and Kuhnlein et a l . (1981) have shown that populations with d i f f e r e n t d i e t s have d i f f e r e n t l e v e l s of f e c a l mutagenicity. Mutagenicity i s considered to be an ind i c a t o r of p o t e n t i a l carcinogenesis since i n v i t r o assays of many substances have shown that most mutagens are carcinogenic, and most car-cinogens are mutagenic (Ames, 1971). As we l l , recent studies have shown that i n d i v i d u a l s from populations at hi g h - r i s k for colon cancer had higher f e c a l mutagenic a c t i v i t y than i n d i v i d u a l s from populations at low-risk for the disease (Reddy et a l . , 1980a; Reddy et a l . , 1980b; Kuhnlein et a l . , 1981). The present study i s an attempt to demonstrate the e f f e c t of dietary modification on f e c a l mutagenic a c t i v i t y . This study was designed to test the hypothesis that short-term con-sumption of d i e t s considered to be rela t e d to high-ri s k for colon cancer . 3 r e s u l t s In s i g n i f i c a n t l y greater mutagenic a c t i v i t y of feces than does consumption of low-risk d i e t s . Six healthy subjects whose habitual d i e t s were omnivorous and n u t r i t i o n a l l y adequate consumed i n sequence baseline d i e t s t y p i c a l of t h e i r own h a b i t u a l d i e t s , experimental lacto-ovo vege-t a r i a n d i e t s thought to be low-risk d i e t s for colon cancer, and h i g h - r i s k d i e t s which were high i n meat and animal f a t and low i n f i b e r . Assays were performed to determine the mutagenic a c t i v i t y of aqueous extracts of f e c a l samples c o l l e c t e d at the end of each d i e t period. Changes i n percent dry weight and pH of the f e c a l samples as w e l l as the number of d a i l y bowel movements on each of the d i e t s were also studied. It was hoped that examination of these parameters might help to explain any changes i n f e c a l mutagenicity as a r e s u l t of dietary modification, or to elucidate the r o l e of d i e t i n the etiology of colon cancer. The short duration of the study - two weeks on the low-risk and high-r i s k d i e t s r e s p e c t i v e l y - and the c o l l e c t i o n of f e c a l samples only at the end of the d i e t periods precluded the opportunity to determine whether an end point for changes i n any of the f e c a l parameters had been reached. Also, the r e l a t i o n s h i p between short-term changes i n f e c a l mutagenicity and changes i n r i s k for colon cancer i s not known. The design of the study did not permit the i d e n t i f i c a t i o n of s p e c i f i c d i e t a r y components responsible for changes i n the f e c a l parameters. However, the study was designed to provide a basis for future research which could lead to reduction, through dietary modification, of the r i s k f or colon cancer. 4 CHAPTER II REVIEW OF THE LITERATURE Organization of the Chapter In t h i s chapter, the r e l a t i o n s h i p between f e c a l mutagens and colon carcinogenesis i s discussed. Studies are presented which support an a s s o c i -ation between di e t and f e c a l mutagenic a c t i v i t y . The components of those theories that are mutually complementary, and might explain the r o l e of d i e t i n the etiology and incidence of colon cancer are discussed. The s p e c i f i c components included i n the discussion are dietary f a t , i n t e s t i n a l m i c r o f l o r a , dietary meat and beef, vegetables and dietary f i b e r . The chapter concludes with a review of the chemical nature of some f e c a l mutagens, and factors which i n h i b i t t h e i r a c t i v i t y . The t h e o r e t i c a l basis of tests for f e c a l mutagens i s discussed. Several types of studies are included i n t h i s review. Epidemio-l o g i c a l studies have been u s e f u l i n implicating d i e t as an important envir- , onmental factor i n colon carcinogenesis. Human c l i n i c a l studies have allowed both comparison of various factors between colon cancer patients and controls, and study of the e f f e c t s of s p e c i f i c d i e t a r y components. Animal studies have provided models which show the capacity of dietary f a t and i t s metabolites to promote carcinogenesis. Laboratory studies using bacteria to test f or the presence of mutagens are also reviewed. Mechanisms to I d e n t i f y Factors Involved i n  Colon Carcinogenesis Mutagenic Properties of Carcinogens The use of demonstrated mutagenicity as an i n d i c a t o r for carcino-g e n i c i t y was i n i t i a t e d by Ames (1971). He assayed numerous compounds, and found that a high proportion of known mutagens were carcinogenic, and a high 5 proportion of known carcinogens were mutagenic. H o l l s t e i n et a l . (1979) also found that carcinogens generally give p o s i t i v e test r e s u l t s i n assays for mutagenicity, and non-carcinogens give negative r e s u l t s . Bridges (1976) reported no clearcut examples of f a l s e p o s i t i v e s among compounds tested. Significance of Fecal Mutagens i n Colon Carcinogenesis Results of tests for mutagenicity using s e n s i t i v e s t r a i n s of bacteria have been extrapolated to i n d i c a t e p o t e n t i a l e f f e c t s i n humans. Ames (1971) supported the v a l i d i t y of t h i s process of extrapolation because mutagens act d i r e c t l y on DNA. He also pointed out that mutagens for higher organisms are generally mutagens for micro-organisms, and vice-versa. The mutagenic a c t i v i t y of human feces has recently been the subject of considerable study since i t i s thought that f e c a l mutagens are r e l a t e d to colon carcinogenesis. Bruce et a l . (1978) revealed the presence of mutagens i n the feces of both normal i n d i v i d u a l s and those with c o l o r e c t a l cancer. Bruce speculated that the mutagen i s of i n t e r n a l o r i g i n since the amount detected i n feces i s probably greater than the amount of the compound i n the d i e t . The amount of the f e c a l mutagen was shown to be increased by increasing dietary f a t and meat (Bruce, 1978). Involvement of the i n t e s t i n a l m i c r o f l o r a i n f e c a l mutagenesis has been hypothesized. Wilkins et a l . (1980) observed that feces with high muta-genic a c t i v i t y had very high mutagenic a c t i v i t y a f t e r anaerobic incubation at 37° C, suggesting b a c t e r i a l production of mutagens over time. Van T a s s e l l et al.(1982) assessed the a b i l i t y of b i l e acids to promote mutagen production i n feces, and found a 2.5 to 5 f o l d increase i n mutagen production a f t e r i n -cubation with b i l e acids i n the feces of subjects who c o n s i s t e n t l y excreted mutagenic feces. They believe that b i l e acids l i k e l y act by s o l u b i l i z i n g precursors for the mutagen which the b a c t e r i a u t i l i z e , and increased presence of b i l e acids may also s e l e c t for increased growth of mutagen-producing 6 b a c t e r i a l species. Mutagenic a c t i v i t y of feces has been correlated with the r i s k f o r colon cancer i n population studies. Reddy et a l . (1980a) examined f e c a l samples from vegetarian Seventh-Day-Adventists from New York and from i n -di v i d u a l s consuming d i e t s high i n f a t and meat. The di e t s for both groups were comparable i n terms of energy and t o t a l protein, but the Seventh-Day-Adventists consumed more f i b e r , fresh vegetables and f r u i t , less t o t a l f a t , more f a t from dairy products, and l e s s protein from meat. No f e c a l samples were immediately mutagenic, but non- Seventh-Day-Adventists showed s i g n i f i -cantly higher 2-acetoaminofluorene induced co-mutagenic a c t i v i t y , i n d i c a t i n g that the f e c a l extracts promoted the e f f e c t of t h i s mutagen. Reddy et a l (1980b) compared f e c a l mutagenic a c t i v i t y i n h i g h - r i s k and low-risk populations for colon cancer. Vegetarian Seventh-Day-Adventists showed no mutagenic a c t i v i t y . In the same study, concentration of f e c a l mutagens as well as the percent of samples with mutagenic a c t i v i t y was found to be higher i n a group of non- Seventh-Day-Adventists from New York compared with i n d i v i d u a l s from Kuopio, Finland. The d i e t s d i f f e r e d i n types and amounts of f i b e r , f a t and protein. Kuhnlein et a l . (1981) also showed that vegetarians, who are considered to be at low-risk for colon cancer by v i r t u e of d i e t , have low l e v e l s of f e c a l mutagens. In a study where subjects consumed e s s e n t i a l l y i d e n t i c a l l i q u i d formula d i e t s , Kuhnlein and Kuhnlein (1980) showed that i n d i v i d u a l s d i f f e r e d i n the l e v e l s of f e c a l mutagens observed, but each i n d i v i d u a l had consistent l e v e l s of f e c a l mutagenicity throughout a study of 47 days or longer. This study indicated that f e c a l mutagenic a c t i v i t y was consistent f o r i n d i v i d u a l s i n the absence of dietary change. The research involving f e c a l mutagens to date provides evidence that f e c a l mutagenic a c t i v i t y i s related to the nature of the d i e t , and that d i e t -ary differences r e s u l t i n differences i n f e c a l mutagenicity. There i s a 7 need for further research to determine i n d i v i d u a l responses to dietary changes. Role of Dietary Fat i n Colon Carcinogenesis Dietary Fat Theory The most comprehensive and widely accepted theory of the environ-mental etiology of colon cancer as a function of d i e t has been defined by H i l l (1974). I t was proposed that a carcinogen i s formed i n s i t u i n the colon by the gut b a c t e r i a l f l o r a from some benign substrate derived from the d i e t . Metabolites of dietary f a t were proposed as the substrates i n t h i s reaction. S p e c i f i c a l l y , the anaerobic bacteria! Zlo&thJAAdm pCLfLapuXytL^-icum was indicated as the primary agent for t h i s reaction. The b a c t e r i a can per-form three of four nuclear dehydrogenation reactions which aromatize the b i l e acid nucleus to y i e l d a substituted cyclopentaphenanthrene which i s known to be carcinogenic ( H i l l , 1974). The substrates selected by H i l l (1974) were b i l e acids. Since t h e i r f e c a l concentration i s re l a t e d to dietary f a t , they are s t r u c t u r a l l y r e l a t e d to the cyclopentaphenanthrenes, and they can be chemically converted into the potent carcinogen 20-methyl-cholanthrene as shown i n Figure 1 ( H i l l et a l . , 1971). H i l l ' s o r i g i n a l hypothesis suggested that nuclear dehydrogenating C l o s t r i d i a produce a carcinogen or co-carcino-gen from b i l e acids. The concentrations of nuclear hydrogenating C l o s t r i d i a and of a v a i l a b l e substrate are d i e t dependent. Considerable evidence s t i l l e x i s t s to support continuing i n t e r e s t i n t h i s theory, and i n p a r t i c u l a r the p o t e n t i a l carcinogenic a c t i v i t y of b i l e acids and neutral steroids. H i l l (1975a) and Renwick (1976) suggested that c h o l e s t e r o l may act as a co-carcinogen promoting the action of carcinogens, and the unsaturated b i l e acids, coprostanol and coprostanone, may act as carcinogens. Cruse et a l . (1979) proposed c h o l e s t e r o l as a co-carcinogen H i l l , 1971 FIGURE 1 Conversion of Deoxycholic Acid to 20-methyl-cholanthrene 9 for human colon career since i t i s an obligatory precursor of b i l e s a l t s . B i l e s a l t s were also hypothesized by Cruse et a l . to have an i n d i r e c t r o l e i n s o l u b i l i z i n g c h o l e s t e r o l , and therefore may f a c i l i t a t e carcinogenesis by maintaining i n t e r a c t i o n between c h o l e s t e r o l and c e l l surfaces i n the lumen. Steroids are s t r u c t u r a l l y s i m i l a r to carcinogenic p o l y c y c l i c aromatic hydrocarbons ( H i l l et a l . , 1971). B i l e acids and neutral steroids can be chemically converted into known carcinogens or co-carcinogens. The conver-sions of c h o l i c a c i d to deoxycholic a c i d , and chenodeoxycholic acid to l i t h o -c h o l i c a c i d i n the presence of 7a-dehydroxylase have been described by Mastromarino et a l . (1976). The a c t i v i t y of t h i s enzyme was increased i n c o l o r e c t a l cancer patients (Mastromarino et a l . , 1976). Deoxycholic acid can be further converted into the potent carcinogen 20-methyl-cholanthrene ( H i l l et a l . , 1971 and H i l l , 1974). Aries et a l . (1970) suggested that b a c t e r i a may be able to perform t h i s conversion. Cholesterol i s converted to coprostanol through an enzymatic reduction of the c h o l e s t e r o l double bond. Coprostanol gives r i s e to coprostanone by the subsequent oxidation of the coprostanol hydroxyl group (Wilkins and Hackman, 1974). Cholesterol dehydro-genase i s the enzyme involved i n these conversions, and was present i n greater concentrations i n the feces of c o l o r e c t a l cancer patients CMastromarino et a l . 1976). Human Epidemiological Studies H i l l et a l . (1971) examined samples of feces representing s i x popu-l a t i o n s i n areas with low and high incidences for colon cancer. The neutral s t e r o i d concentration i n feces from low incidence areas was very low. Copros-tanol and coprostanone, the b a c t e r i a l neutral s t e r o i d metabolites of cholest-e r o l , constituted 43% to 62% of the t o t a l neutral s t e r o i d s compared with 64% to 71% i n thehhighincidence populations. Similar r e s u l t s were obtained by Wynder and Reddy (1974) who reported that the d a i l y f e c a l excretion of c h o l e s t e r o l metabolites, and the r a t i o of coprostanol and coprostanone to c h o l e s t e r o l were s i g n i f i c a n t l y elevated (p<0.01) i n Americans on a mixed western di e t as compared to other populations considered at low r i s k . Fine-gold et a l . (1974) also found that subjects from countries with a high incidence of colon cancer had elevated concentrations of acid steroids i n feces, and a greater percentage of these steroids were present i n the degraded form. Therefore, the gut f l o r a i n high r i s k populations must have a potent enzyme to carry out the degradation ( H i l l , 1975a). Reddy (1981a) reviewed several epidemiological studies which ind i c a t e ani'Mpprtant r e l a t i o n s h i p between dietary f a t and production of b i l e acids i n the etiology of colon cancer. Animal Studies A number of animal studies have served to document the r o l e of b i l e acids and neutral steroids as co-carcinogens or promoters of colon carcino-genesis. Although the mechanism has not been elucidated, Reddy (1981b) reported on animal studies which indicated that l i t h o c h o l i c acid, tauro-deoxycholic acid and deoxycholic acid act as promoters of known carcinogens. Reddy et a l . (1977a) showed that r a t s fed d i e t s containing 20% corn o i l or l a r d excreted larger amounts of b i l e acids of b i l i a r y o r i g i n , and larger amounts of b i l e acids and neutral steroids of dietary o r i g i n than those fed di e t s containing 5% f a t . Subsequent treatment with the carcinogen 1,2-di-methyl-hydrazine resulted i n increased colon tumors on the 20% f a t d i e t . Other studies confirmed that high f a t d i e t s increased the suscepti-b i l i t y to colon cancer induction by model carcinogens i n laboratory animals. Reddy et a l . (1977b) showed that rats fed a 20% f a t d i e t exhibited an increased incidence of colon tumors when the carcinogens 1,2-dimethyl-hydrazine, methylazoxymethanol acetate or methylnitrosurea were administered. Similar r e s u l t s were described by B u l l et a l . (1979) when i n t e s t i n a l cancer 11 induced by azoxymethane was promoted by a high (.30%) f a t d i e t fed a f t e r the administration of azoxymethane. The e f f e c t s were observed even with a delay of 10 weeks between the administration of azoxymethane and the high f a t d i e t . A study by Nigro et a l . (1976) also showed i n t e r r e l a t i o n s h i p s among dietary f a t , f e c a l b i l e acids, and the administration of azoxymethane. The administration of a high f a t diet resulted i n increased excretion of t o t a l f e c a l neutral steroids, and administration of the carcinogen increased the degradation of c h o l e s t e r o l which i n turn may enhance the promotion of c a r c i n -ogenesis. Another study by Nigro (1981) showed that when cholestyramine, a drug known to increase f e c a l b i l e acids, was fed to r a t s given azoxymethane, there was an increase i n the development of tumors i n the d i s t a l large i n t e s t i n e . Animal studies also provide evidence that b i l e acids are capable of producing tumors at the s i t e of i n j e c t i o n . Wynder and Reddy (1974) observed that the b i l e acids deoxycholic acid, l i t h o c h o l i c acid, bisnor-5-cholenic acid and apocholic acid produce tumors following i n j e c t i o n . These experimental observations regarding the r o l e of d i e t and excessive f e c a l steroids i n animal models tend to support the findings from human studies which ind i c a t e an a s s o c i a t i o n between high l e v e l s of f e c a l steroids and increased r i s k for colon cancer (Nigro, 1981). Evidence Against the Role of Dietary Fat i n Colon Carcinogenesis Although there i s supportive evidence for the theory defined by H i l l (1974), there i s also some evidence to the contrary. Mower et a l . (1979) examined b i l e a c i d concentrations i n high- and low-risk Japanese populations, and found greater concentrations of deoxycholic acid but l e s s e r concentrations of other b i l e acids i n the feces of the h i g h - r i s k population. In another study, the intake of western foods was not p o s i t i v e l y correlated with the f e c a l degraded b i l e acids l i t h o c h o l i c and deoxycholic acids derived from 12 c h o l i c and chenodeoxycholic acids by b a c t e r i a l action. (Mower et a l . , 1978). Rather, the consumption of two Japanese food items, pickled turnip and p i c k l e d plum, correlated negatively with modified b i l e acids i n feces, and may con-t r i b u t e to lower concentrations of b i l e acids. There was no c o r r e l a t i o n between presence of undegraded b i l e acids i n feces and the consumption of Japanese foods. Moskovitz et a l . (1979)'reported s i m i l a r excretion of b i l e acids and steroids i n the feces of colon cancer patients, patients with non-gastro-i n t e s t i n a l cancers, and normal subjects. In f a c t , the group of colon cancer patients excreted lower amounts of b a c t e r i a l l y transformed s t e r o i d s . This f i n d i n g also contradicts H i l l ' s theory. However, the intakes of dietary fa t were not compared i n t h i s study. Role of I n t e s t i n a l M i c r o f l o r a i n Colon Carcinogenesis Human Epidemiological Studies B i l e s a l t deconjugation, an e s s e n t i a l f i r s t step permitting further degration of b i l e s a l t s , can be c a r r i e d out by most s t r a i n s of anaerobic b a c t e r i a present i n feces — bacteroides, b i f i d o b a c t e r i a , eubacteria, C l o s t -r i d i a , v e i l l o n e l l a and enterococci ( H i l l , 1975a). Epidemiological and pop-u l a t i o n studies support the r o l e of b a c t e r i a i n the etiology of colon cancer by showing quantitative differences among population groups i n groups of f e c a l b a c t e r i a . H i l l et a l . (1971) examined f e c a l b a c t e r i a i n i n d i v i d u a l s i n high and low incidence areas for colon cancer. England, Scotland and the U.S.A. were the high incidence areas, and were compared with Uganda, Japan and India. The same broad groups of b a c t e r i a were found i n the feces from a l l of the s i x populations, but there were s u b s t a n t i a l differences i n r e l a t i v e numbers of several b a c t e r i a l groups. The B r i t i s h and American subjects yielded many more anaerobic bacteroides species, while the low incidence populations had many more aerobic bacteria. The r a t i o of anaerobes 'to aerobes was much higher i n the people l i v i n g on a western diet than i n those on l a r g e l y vegetarian d i e t s . These r e s u l t s were found to be inde-pendent of race or climate. Aries et a l . (1969) also recorded the presence of more anaerobic b a c t e r i a i n the feces of populations with high colon cancer rates. A study by H i l l et a l . (1975) showed an.increase i n nuclear dehydro-genating C l o s t r i d i a i n the feces of patients with diagnosed large bowel cancer over patients with other types of cancer. This study also provided evidence to support the r o l e of some groups of i n t e s t i n a l b a c t e r i a i n carcinogenesis. E f f e c t s of Diet on I n t e s t i n a l M i c r o f l o r a In s p i t e of the b e l i e f that b a c t e r i a have a r o l e i n human colon cancer, s o l i d evidence does not yet e x i s t . Studies on b a c t e r i a l f l o r a have f a i l e d to demonstrate e f f e c t s of changing various components of the d i e t on the composition of f e c a l f l o r a . Wilkins et a l . (1980) found that i n d i v i d u a l s i n the same population and consuming s i m i l a r d i e t s have very d i f f e r e n t b a c t e r i a l f l o r a s . In another study by Cummings et a l . (1978), changes i n dietary f a t composition did not change the number of b a c t e r i a l groups observed. The e f f e c t s of meat consumption on f e c a l m i c r o f l o r a have been studied (Cummings et a l . , 1979; Maier et a l . , 1974; Hentges et a l . , 1977) and found not to be s i g n i f i c a n t . Consumption of two types of dietary f i b e r , bran and bagasse, was also found to have no e f f e c t on f e c a l m i c r o f l o r a (Baird et a l . , 1977; Walters et a l . , 1975; Cummings et a l . , 1976). H i l l (1981) suggested that detectable changes i n f l o r a might occur over.longer term experiments of up to 12 months. One study by Crowther et a l . (1973) showed a reduction i n anaerobic and some components of aerobic f e c a l f l o r a i n subjects fed a l i q u i d , chemically formulated, residue f r e e d i e t . Johnson (1980) speculated that the colon of each person may be populated by b a c t e r i a l s t r a i n s s p e c i f i c f o r that i n d i v i d u a l , and found that there may be one or several s t r a i n s present of each species of b a c t e r i a . Hentges et a l . (1977) also found v a r i a t i o n s i n f e c a l f l o r a among i n d i v i d u a l s , but maintained that b a c t e r i a l a c t i v i t y may be important regardless of species. Enzymatic A c t i v i t y of the I n t e s t i n a l M i c r o f l o r a Some evidence suggests that changes i n the composition of d i e t s do not change the major groups of colonic b a c t e r i a , but rather a l t e r the meta-b o l i c a c t i v i t y of colonic b a c t e r i a as evidenced by changes i n the a c t i v i t y of key enzymes (Bornside, 1978). The presence, concentration and a c t i v i t y of various enzymes i n feces have been investigated as i n d i c a t o r s associated with f e c a l m i c r o f l o r a and the r i s k for colon cancer. Enzymes which have been r e l a t e d to increased r i s k for colon cancer are g-glucuronidase, chole-s t e r o l dehydrogenase, 7a-dehydroxylase and 7a~dehydrogenase (Reddy, 1981a). A b a c t e r i a l enzyme which releases active carcinogen metabolites by the hydrolysis of glucuronide conjugates i n the lumen of the gut i s g-glu-curonidase (Reddy et a l . , 1977c). Goldin and Gorbach (1976) discussed the importance of t h i s reaction whereby many substances which are d e t o x i f i e d i n the l i v e r enter the bowel v i a b i l e i n the form of glucuronides, and could be regenerated i n s i t u i n the bowel. Reddy et a l . (1974) measured g-glucuronidase a c t i v i t y i n the feces of subjects who consumed 454 g d a i l y of beef, pork or chicken, and l a t e r were transferred to a non-meat d i e t . The a c t i v i t y of the enzyme was con-siderably higher while the high meat di e t was being consumed. They con-cluded that the i n t e s t i n a l m i c r o f l o r a of i n d i v i d u a l s on a high meat d i e t have an increased capacity to hydrolyze glucuronic acid conjugates, thus increasing the l i k e l i h o o d for carcinogens or other toxic metabolites to be released. Reddy et a l . (1980c) observed a s i m i l a r r e s u l t , and further showed that reverting the enzyme a c t i v i t y back when the high meat diet was discontinued was a slower process. Animal studies showed that the a c t i v i t y of g-glucuronidase was increased when a high beef d i e t was fed (Goldin and Gorbach, 1976), and when dietary protein and f a t were increased (Reddy et a 1980c). Goldin and Gorbach also noted that the enzyme a c t i v i t y took 20 day to reach the peak, suggesting that changes i n f e c a l b a c t e r i a l f l o r a do occur with time. Cholesterol dehydrogenase i s involved i n the reduction of the chole s t e r o l double bond and subsequent oxidation of c h o l e s t e r o l to coprostanol and coprostanone. Wilkins and Hackman (1974) observed two groups within a high- r i s k population for colon cancer. Low conversion of c h o l e s t e r o l was observed i n 25% of the i n d i v i d u a l s studied, and 75% showed high conversion. Whether these two groups d i f f e r e d i n t h e i r r i s k f or colon cancer i s not known. However, Mastromarino et a l . (1976) examined the a c t i v i t y of c h o l e s t e r o l dehydrogenase i n the feces of cancer patients, patients with adenomatous polyps, and normal controls. The colon cancer patients had the highest l e v e l s of the enzyme. The enzymes7a-dehydroxylase and 7a-dehydrogenase are r e s p e c t i v e l y capable of the reduction and oxidation of b i l e s a l t s to y i e l d l i t h o c h o l i c a c i d , deoxycholic acid, or t h e i r keto d e r i v a t i v e s (Kay, 1981). Studies by Mastromarino et a l . (1976) and Macdonald et a l . (1978) have demonstrated an increased a c t i v i t y i n these enzymes i n colon cancer patients over normal controls and i n omnivores over vegetarians. Pomare and Heaton (1973) demonstrated that formation of hydroxylated b i l e s a l t s i n the b i l e of femal subjects decreased when unprocessed bran was added to the d i e t , and postu-lated i n h i b i t i o n of 7 a-dehydroxylase. Nitroreductase and azoreductase are other enzymes which have been shown to have increased a c t i v i t y i n feces when a high beef d i e t was fed (Goldin and Gorbach, 1976). These enzymes are responsible for reducing n i t r o - and azo- compounds to aromatic amines. Both the intermediates and 16 end products are p o t e n t i a l mutagens and carcinogens. This reaction may be s i g n i f i c a n t since such compounds are sometimes used as additives i n the west-ern d i e t (Goldin and Gorbach, 1976). Role of Dietary Components i n Colon Carcinogenesis E f f e c t s of Meat and Beef on Colon Carcinogenesis While epidemiological studies i n d i c a t e an association between t o t a l f a t consumption and the incidence of colon cancer (Wynder and Shigematsu, 1967; Wynder, 1979), i t has been recognized that a major portion of dietary f a t comes from meat. In p a r t i c u l a r , beef contributes s i g n i f i c a n t l y to over-a l l f a t intake (Reddy et a l . , 1975) and has therefore been implicated i n the etiology of the disease. Maier et a l . (1974) reported on the e f f e c t s of "normal", meatless and high meat d i e t s on ten male subjects. Analysis of f e c a l samples c o l l e c t e d during the l a s t week on each di e t regimen showed increased coliforms on the meatless d i e t , and increased anaerobic bacteroides on the high meat d i e t , although the numbers were not s i g n i f i c a n t . Reddy et a l . (1974) showed that subjects who transferred from a high meat, high f a t d i e t to a meatless diet had s i g n i f i c a n t l y fewer anaerobic b a c t e r i a i n feces. Hentges et a l . (1977) observed a s i m i l a r but not s i g n i f i c a n t s h i f t i n the r a t i o of anaerobes to aerobes on a high beef d i e t . In a study by Reddy et a l . (1980c), assays for ^-glucuronidase, 7a~dehydroxylase and c h o l e s t e r o l dehydrogenase were performed on f e c a l samples obtained from subjects who consumed d i e t s high i n rare, medium or well-done beef over a four week period. The degree to which the beef was cooked had no s i g n i f i c a n t e f f e c t . However, ^-glucuronidase a c t i v i t y was s i g n i f i c a n t l y higher when the subjects consumed the high f a t , high beef experimental d i e t s i n comparison with t h e i r customary d i e t s . Changes i n the f e c a l excretion of b i l e acids and steroids r e l a t e d to the consumption of a high meat test d i e t have been reported by Reddy et a l . (1974). A s i g n i f i c a n t increase i n the excretion of m i c r o b i a l l y modified b i l e acids was observed. Cummings et a l . (1979) also reported a small increase i n b i l e acid excretion when subjects transferred from a di e t low i n meat pr o t e i n to a high meat protein d i e t . Reddy et a l . (1980c) showed higher f e c a l excretion l e v e l s of the c h o l e s t e r o l metabolites, coprostanol and coprostanone, as well as the secondary b i l e acids, deoxycholic and l i t h o c h o l i c acids, when subjects consumed a high f a t , high beef d i e t . Decreased f e c a l wet weight or percent wet weight, or increased t r a n s i t time have not been observed r e l a t e d to a high f a t , high beef d i e t (Reddy et a l . , 1980c; Cummings et a l . , 1979). However, the f i b e r content of the d i e t s was not con t r o l l e d i n these studies. : In an animal study by Nigro et a l . (1975), r a t s fed a high beef di e t exhibited enhanced number and s i z e of i n t e s t i n a l tumors when azoxy-methane was administered. This shows a promotional e f f e c t of dietary beef i n carcinogenesis. An alternate r o l e f o r beef i n mutagenesis has been proposed by Commoner et a l . (1978) i n the report on a study of cooking procedures. Mutagenic a c t i v i t y of well-done beef was re l a t e d to the formation of benzo (a) pyrene during the cooking. The study also indicated the production of mutagenic substances when beef stock i s heated and condensed, or during the cooking of ground beef at temperatures below 200° C. These substances were not benzo (a) pyrene or the p y r o l y s i s products of amino acids or protein. The formation of mutagens i n f r i e d or b r o i l e d beef was reported by deVet (1981). The r o l e of beef i n the etio l o g y of colon cancer was refuted by Sherlock (1980) when he reported that the incidence of c o l o r e c t a l cancer i n Mormons i n Utah remained stable over a 25 year period during which the consumption of beef increased s i g n i f i c a n t l y . 18 E f f e c t s of Dietary Fat from Non-meat Sources on Colon Carcinogenesis Recent studies on Finns who are a low incidence population f o r colon cancer, suggested that the type of dietary f a t consumed may have more s i g n i -ficance than the amount. Reddy et a l . (1978) reported that t h i s population has a high dietary intake of f a t from dairy products, and a meat intake lower than Americans who are a high incidence population. Analysis of feces from both populations showed a lower concentration of b i l e acids i n the Finnish group. However, Reddy (1981a) reported on animal studies comparing the e f f e c t s of various types of f a t i n promoting tumorigenesis, and sug-gested that t o t a l dietary f a t should be considered. Epidemiological studies c r i t i q u e d by Enig et a l . (1978) provide some support f o r the implication of vegetable f a t as a promotor of carcinogenesis. The mechanism proposed was that processed vegetable f a t s , e s p e c i a l l y trans f a t t y acids, may a l t e r the permeability of c e l l membranes to carcinogens. E f f e c t s of Vegetables on Colon Carcinogenesis The consumption of c e r t a i n vegetables has been associated with a lower r i s k f o r cancer of the colon and rectum. Graham et a l . (1978) compared the di e t s of patients with colon cancer, r e c t a l cancer, and other types of cancers. Higher l e v e l s of consumption of vegetables, e s p e c i a l l y raw vege-tables, was re l a t e d to a lower r i s k f o r cancer of the colon and rectum. Con-sumption of cabbage and related cruciferous vegetables has also been rel a t e d to a lower incidence of colon cancer, although the mechanism i s not known (Graham and M e t t l i n , 1979). Role of Dietary Fiber i n Colon Carcinogenesis Fiber Theory The c o n t r o v e r s i a l theory on the protective r o l e of dietary f i b e r i n the etiology of colon cancer was pioneered by B u r k i t t , 1969; B u r k i t t , 1971; B u r k i t t , 1974; Burkitt et a l . , 1974). He supposed that d i e t -ary f i b e r protects by decreasing t r a n s i t time and increasing bulk of feces, thus minimizing the contact of carcinogens with the i n t e s t i n a l mucosa. Burk i t t thought that a deficiency i n dietary f i b e r i s a common causative factor i n the epidemiology of colon cancer and other non-infective gastro-i n t e s t i n a l and c i r c u l a t o r y diseases. The epidemiology of colon cancer was perceived to be markedly s i m i l a r to that of coronary artery disease, append-i c i t i s , d i v e r t i c u l a r disease, varicose veins, hiatus hernia and hemorrhoids. Examination of dietary changes taking place i n the western world i n r e l a t i o n to the pattern of these diseases supported the i m p l i c a t i o n of the consumption of a refin e d d i e t as a causative factor f o r colon cancer (Burkitt, 1973; Walker and B u r k i t t , 1976). The p r i n c i p a l d i etary changes i d e n t i f i e d were increases i n the consumption of animal protein, sugar, f a t and r e f i n e d cereals, and a decrease i n the intake of crude f i b e r , p a r t i c u l -a r l y i n cereal foods. Burkitt et a l . (1974). postulated that i f the environment of the large bowel mucosa i s dominated by the f e c a l content, then dietary f i b e r i s important i n that i t i s not digested or absorbed but reaches the large bowel unchanged. Further, Burkitt's work with black Africans (Burkitt, 1971) led him to conclude that a d i e t high i n vegetable f i b e r r e s u l t i n g i n high f e c a l output was responsible for that population's freedom from colonic disorders. His theory supposed that the minimally processed d i e t consumed by black Africans and other low-risk populations contained adequate f i b e r , and was associated with i n t e s t i n a l b a c t e r i a l populations and b i l e s a l t concentrations which would beconsidered 'normal'. Few, i f any, p o t e n t i a l carcinogens would be present, and a short contact time between a d i l u t e and bulky s t o o l and the large bowel mucosa would r e s u l t i n a low incidence of large bowel tumors. In contrast, the highly refined western d i e t , ' d e f i c i e n t ' i n f i b e r content, led to an a l t e r e d population of bacteria i n the large i n t e s t i n e which would be capable of degrading b i l e s a l t s to form carcinogens. Constipation was con-sidered to be the manifestation of a f i b e r d e f i c i e n t d i e t , and prolonged contact time between a concentrated s t o o l and the i n t e s t i n a l mucosa would allow the development of large bowel tumors. Bu r k i t t ' s diagramatic repres-entation of the postulated r e l a t i o n s h i p between d i e t and cancer of the large bowel i s presented i n Figure 2. Much consideration has been given to the r o l e of f i b e r i n the d i e t i n r e l a t i o n to B u r k i t t ' s theory. Although a generally accepted d e f i n i t i o n of dietary f i b e r has not been achieved (Talbot, 1981), Trowell (1976) defined dietary f i b e r as the residue of plant food r e s i s t a n t to hydrolysis by human alimentary enzymes. Dietary f i b e r includes c e l l u l o s e , hemicellulose, pectin, l i g n i n , c u t i n and gum (Huang et a l . , 1978; Upton, 1980). Macdonald and Rao (1979) emphasized that dietary f i b e r i s not a s i n g l e substance, but a mixture of complex polymers which vary i n t h e i r a b i l i t y to absorb water and b i l e salts, i n t h e i r r e l a t i v e d i g e s t i b i l i t y , and i n t h e i r bulking action i n the gut. The r e s u l t s of studies on dietary f i b e r i n d i c a t e that further research on the e f f e c t s of i n d i v i d u a l f i b e r types and components i s needed. E f f e c t s of Fiber on Bowel Transit Time The assumption that increased dietary f i b e r shortens i n d i v i d u a l bowel t r a n s i t time has not been conclusively substantiated. A long t r a n s i t time has been hypothesized to increase contact time between carcinogens and the i n t e s t i n a l lumen, hence increasing the r i s k for colon cancer (Burkitt, 1971). Although the r e s u l t s of a large study on comparative t r a n s i t times of popu-l a t i o n s at low- and h i g h - r i s k for colon cancer were interpreted to show that the presence of f i b e r shortens t r a n s i t time (Burkitt, et a l . , 1972), other studies suggested that t r a n s i t time was not related to the pathogenesis of colonic disease. Wynder and Shigematsu (1967) conducted a retrospective study on patients with cancer of the colon, and concluded that any a s s o c i -FOOD Little processed Adequate fibre No surplus Highly processed refined carbohydrate 1 1 Normaj intestinal bacteria Bile salts normal I-Short contact between diluted stool content and mucosa Little or no carcinogens Tumours rare Deficient fibre Surplus refined carbohydrate 1 Altered bacterial content Degradation of bile salts 1 Prolonged contact between concentrated stool content and mucosa Carcinogens Tumours common Burkitt, 1971 FIGURE 2 Postulated Relationship Between Dietary Fiber and Colon Cancer ation between constipation and colon cancer was l i k e l y to be i n c i d e n t a l to obesity. Glober et a l (1974) measured t r a n s i t times i n Japanese migrants to Hawaii and i n Caucasians. The r e s u l t s of the study showed that although Japanese migrants develop disease patterns s i m i l a r to Caucasians, t r a n s i t times remained s i m i l a r to those observed i n non-migrant Japanese, even i n second generation migrants. The conclusion was that t r a n s i t time did not appear to r e l a t e to the epidemiology of colon cancer. On the basis of studies reviewed, Walker (1976) and Mendeloff (1976) maintained that there was no c o r r e l a t i o n between constipation and cancer. Conversely, Cleveland and Cole (1969) found no e f f e c t on the chemical induction of colon tumors i n rodents when laxatives were fed. Further, the addition of f i b e r to the d i e t i n the form of c e l l u l o s e or bran did not have an e f f e c t on the f e c a l t r a n s i t time of i n d i v i d u a l s (Eastwood et a l . , 1973a; Eastwood et a l . , 1973b). In a study of s i x sub-j e c t s , Beyer and Flynn (1978) recorded a change i n t r a n s i t time between low and high f i b e r d i e t s , but paired observations for i n d i v i d u a l subjects on each of the d i e t s were not a v a i l a b l e . E f f e c t s of Fiber on Fecal Bulk The r o l e of f i b e r i n increasing the bulk of feces has been generally supported. The capacity of f i b e r to take up and hold water i s believed to be important, although the exact mechanism of f e c a l bulking i s unknown. Studies where the ingestion of crude f i b e r , bran, c e l l u l o s e or bagasse was increased have demonstrated a s t o o l bulking e f f e c t (Eastwood et a l . , 1973a; Eastwood et a l . , 1973b; Fuchs et a l . , 1976; Baird et a l . , 1977). However, Stephen and Cummings (1979) compared the e f f e c t s of various f i b e r preparations on f e c a l weight and water holding capacity, and concluded that those pre-parations with a greater water holding capacity had l e s s e f f e c t on f e c a l bulk. This r e s u l t i s the reverse of general expectation, and the implications are unclear, except that the assessment of the r o l e of f i b e r i s s t i l l incomplete. E f f e c t s of Fiber Type on Fecal B i l e Acids Various studies have examined the e f f e c t s of sources of f i b e r on the amount and concentration of b i l e acids excreted i n feces. In three studies where bran was added to the d i e t , there was no observed e f f e c t on the t o t a l excretion of b i l e acids i n feces (Eastwood, 1973a; Eastwood et a l . , 1973b; Baird et a l . , 1977). However, d i l u t i o n of b i l e acids (Eastwood et a l . , 1973a) and neutral steroids (Walters et a l . , 1975) were observed. Pomare and Heaton (1973) further showed that dietary bran reduced the amount of dehydroxylated b i l e sales i n b i l e . When c e l l u l o s e was added to the d i e t , the concentration of b i l e acids i n feces was decreased, but c o n f l i c t i n g r e s u l t s were obtained for the e f f e c t on t o t a l b i l e acid excretion (Eastwood et a l . , 1973a; Eastwood et a l . , 1973b). The addition of bagasse was shown to stimulate the t o t a l loss of f e c a l b i l e acids, although the concentration i n the feces, remained unchanged Baird et a l . , 1977; Walters et a l . , 1975). Although Burkitt (1971) contended that cereal f i b e r i s superior i n i t s a b i l i t y to protect against colon cancer, the lack of general agreement among these studies suggests the need to further examine the r o l e s of various types of f i b e r (Freeman, 1979; Huang et a l . , 1978). In addition, i t has been suggested that i n d i g e s t i b l e protein could also play a s i g n i f i c a n t r o l e i n the observed e f f e c t s of dietary fiber, (Saunders, 1980). Evidence Against the Fiber Theory There have been several research reports which apparently contradict B u r k i t t ' s f i b e r hypothesis. Fuchs et a l . (1976) examined the e f f e c t s of crude f i b e r supplementation on human f e c a l m i c r o f l o r a , and observed an i n -creased r a t i o of anaerobic to aerobic b a c t e r i a , and an increase i n the t o t a l 24-hour f e c a l excretion of anaerobic b a c t e r i a . A group of Seventh-Day-Adventists i n Loma Linda, C a l i f o r n i a had a low incidence of colon cancer (Lemon et a l . , 1946), but consumed di e t s low i n crude f i b e r . In the Canadian A r c t i c , some groups of Inuit who were reported to consume d i e t s low i n plant f i b e r were not n e c e s s a r i l y at h i g h - r i s k for colon cancer (Schaefer, 1959). Haenszel et a l . (1973) i n a case-control study found that Hawaiian Japanese cancer patients more frequently ingested f i b e r - c o n t a i n i n g legumes. Chemical Nature of Fecal Mutagens, and I n h i b i t o r s Possible Role for Nitroso-type Carcinogens Although the evidence favors the metabolites of b i l e acids as the main e t i o l o g i c a l agents of colon cancer ( H i l l , 1980), some consideration has been given to the p o t e n t i a l r o l e of nitroso-type carcinogens. Bruce et a l . (1978) reported the presence of mutagenic N-nitroso compounds of endogenous o r i g i n i n human feces. Rao et a l . (.1981) showed that f e c a l mutagenic a c t i v i t y increased when sodium n i t r i t e was incubated with human feces. They hypo-thesized that formation of v o l a t i l e N-nitroso compounds was responsible for the increased mutagenicity. I n t e r n a l l y formed N-nitroso compounds can be produced by the acid catalyzed reaction of n i t r i t e , formed by b a c t e r i a l r e -duction of dietary n i t r i t e s and secondary amines with s u i t a b l e nitrogen com-pounds (Mirvish, 1975; H i l l , 1980; Issenberg, 1976). H i l l further suggested that the products of t h i s reaction required a c t i v a t i o n to give the ultimate potent carcinogen. Mandel et a l . (.1977) reported that nitrosamides which react d i r e c t l y with DNA without metabolic a c t i v a t i o n by l i v e r microsomal enzymes, can be formed by the transfer of a n i t r o s o group from a nitrosamine to amide as shown i n Figure 3. This n i t r o s o exchange reaction i s catalyzed by enzymes present i n c e r t a i n i n t e s t i n a l b a c t e r i a . Dietary n i t r a t e i s u n l i k e l y to be an important f a c t o r i n the pro-duction of nitroso-type carcinogens since most i s probably absorbed undegraded from the upper small i n t e s t i n e ( H i l l , 1975b). Evidence for the endogenous 25 N = 0 ^ H O BACTERIAL w ^-fi—O I + I II • EXCHANGE W _ ' R - N —R R - N - C - R ENZYME R - N - C — R Nitrosamine Amide Nitrosamide FIGURE 3 The Nitroso Exchange Reaction synthesis of n i t r i t e and n i t r a t e i n humans was provided by Tannenbaum et a l . (.1978). The l e v e l s of urinary excretion of n i t r a t e i n six subjects on pro-t e i n free and low protein d i e t s exceeded the dietary intake, and indicated an endogenous source. Subsequent examination of f e c a l samples from normal subjects and ileostomy exudate samples showed the presence of n i t r i t e i n a l l of the f e c a l and ileostomy samples, and n i t r a t e i n the f e c a l samples ex c l u s i v e l y . The implications of these studies were i n favor of n i t r i t e formation by the n i t r i f i c a t i o n of ammonia or organic nitrogen (Wrong, 1978) i n the upper aerobic portion of the i n t e s t i n e , and the formation of n i t r a t e by b a c t e r i a l action i n the e s s e n t i a l l y anaerobic large i n t e s t i n e . Suzuki and Mitsuoka (1981) also found v o l a t i l e nitrosamines i n feces, which they hypothesized to be formed i n the i n t e s t i n e by i n t e s t i n a l b a c t e r i a . They found that a high protein, high f a t diet increases the output of the f e c a l nitrosamines. The l e v e l of v o l a t i l e n i t r o s o compounds i n feces, although s i m i l a r to some foods, may pose a greater hazard since the compounds may be absorbed into the blood and urine (Wang et a l . , 1978). Three secondary amines which may be involved i n the sequence of formation of N-nitroso compounds are thought to be produced i n the large bowel. Dimethylamine from l e c i t h i n or choline, piperadine from l y s i n e , and p y r r o l i d i n e from arginine or ornithine are p o t e n t i a l l y n i t r o s a t a b l e by the gut f l o r a at neutral pH i n the presence of adequate n i t r a t e or n i t r i t e ( H i l l , 1975a; H i l l and Drasar, 1974; Wang et a l . , 1978; Issenberg, 1976). Recent research by Lee et a l . (1981) using improved analysis tech-niques for v o l a t i l e nitrosamines i n feces suggests that the concentration of these compounds i s extremely low, and that the nitrosamines are r e l a t i v e l y i n e r t i n the colon. However, they do not completely r u l e out the p o s s i b i l i t y of nitrosamine formation under aerobic conditions near the i n t e s t i n a l w a l l , or i n the upper g a s t r o i n t e s t i n a l t r a c t . 27 I n h i b i t o r s of Mutagenesis A possible r o l e for Vitamin C as an anticarcinogen which acts by i n h i b i t i n g nitrosamine formation i n the gut has been proposed (Upton, 1980). The vitamin C competes for n i t r i t e , and lessens the n i t r o s a t i o n process (Mirvish, 1975). Land and Bruce (1978) observed that the concentration of N-nitroso mutagens i n feces was depressed by 48% when 4 g supplementary vitamin C was ingested d a i l y . The unsaturated f a t t y acids o l e i c acid and l i n o l e i c a c i d i n f e c a l extract have also been shown by Hayatsu et a l . (1981) to i n h i b i t the a c t i v i t y of known mutagens. A s i m i l a r e f f e c t was not observed when saturated f a t t y acids were tested for capacity to i n h i b i t mutagenic a c t i v i t y . The mechanism for i n h i b i t i o n has not been confirmed, but p h y s i c a l trapping of the mutagens was hypothesized (Hayatsu, et a l . , 1981). Tests for Fecal Mutagens Sensitive b a c t e r i a l t e s t e r s t r a i n s have frequently been used to detect the presence of mutagens i n v i t r o . The b a c t e r i a SalmoYlzZlOL typhimvJUJUm wild type can synthesize a l l i t s e s s e n t i a l amino acids from inorganic nitrogen (Venitt, 1980). Ames, et a l . (1975) developed mutant s t r a i n s of t h i s bact-erium species which require the amino acid h i s t i d i n e i n the growth medium. They are lipopolysaccharide d e f i c i e n t , and thus more permeable to the passage of large molecules such as mutagens. Some of these tester s t r a i n s also have a deleted excision repair system, and are much more s e n s i t i v e to various mutagens (Ames et a l . , 1973a). Two commonly used tester s t r a i n s of SdlmonelZa, typhimuAAJUm are TA 100 and TA 98. TA 100 i s s e n s i t i v e to mutagens which cause base-pair s u b s t i t u t i o n s , and TA 98 i s s e n s i t i v e to mutagens which cause frameshift mutations, or s h i f t e d p a i r i n g i n r e p e t i t i v e sequences of DNA, which leads to addition or d e l e t i o n of base pairs i n the DNA sequence (Ames et a l . , 1975; Ames et a l . , 1973b). Such damage to DNA r e s u l t s i n a 28 mutation causing a change i n phenotype of the b a c t e r i a . They are reverted from requiring h i s t i d i n e i n the growth medium back to the h i s t i d i n e independ-ent wild type by a v a r i e t y of mutagens. Venitt (1980) defined mutation as a stable, h e r i t a b l e change i n a DNA nucleotide sequence, which may be detected as a phenotypic change. The "Ames t e s t " (Ames, et a l . , 1975) measures the frequency of a reverse mutation i n ScUbnoneZZa typhAmuJvumto test f or the presence of mutagens. H i s t i d i n e requiring b a c t e r i a grow to exhaust the small amount of h i s t i d i n e present i n a h i s t i d i n e l i m i t e d growth medium, a f t e r which only revertant b a c t e r i a , with the capacity to synthesize h i s t i d i n e , can continue to grow (Green, et a l . , 1976). Various modifications of the Ames system have been used to measure the mutagenic a c t i v i t y of human f e c a l extracts, Bruce et a l . (1978) reported that c e r t a i n f e c a l samples contained mutagens which do not need a c t i v a t i o n using enzymes obtained from the induced tissues of animals, usually rat l i v e r , and which act on TA 100. Ehrich et a l . (1979) confirmed that human f e c a l extracts can be mutagenic for TA 98. Bruce and Dion (1980) used the Ames test to show that populations with a high incidence of colon cancer have a higher propensity towards mutagenic a c t i v i t y of feces. Nader et a l . (1981) measured the mutagenic a c t i v i t y of feces from i n d i v i d u a l s at high-r i s k and low^-risk for colon cancer using the Ames t e s t . Bruce et a l . (1978) reported on the use of the Ames test to i d e n t i f y nitrosamines as a group of di e t dependent compounds of i n t e r n a l o r i g i n responsible for base pair sub-s t i t u t i o n mutagenicity i n f e c a l extracts. Various methods of preparation of f e c a l extracts for mutagen t e s t i n g have been reported i n the l i t e r a t u r e . Ether, ethanol, dimethyl sulfoxide and water have a l l been used for preparing extracts. Metabolic a c t i v a t i o n using mammalian enzyme preprations (Ames et a l . , 1975) has also been incon-s i s t e n t . Therefore, i t i s l i k e l y that there are a v a r i e t y of compounds which 29 account f o r the mutagenic a c t i v i t y observed throughout these experiments. In addition, Bruce et a l . (1978) showed that ether extracts appeared to contain i n h i b i t o r s of mutagenicity, as w e l l as substances toxic to test organisms. Lu r i a and Delbruck (1943) described a f l u c t u a t i o n test which had the capacity to detect mutagens at very low concentrations. The test requires that the i n i t i a l numbers of bac t e r i a used i n the test to detect mutagens be small enough f o r the number of mutation events during the f i r s t d i v i s i o n cycle of the b a c t e r i a to be measureable. H o l l s t e i n et a l . (1979) i n a r e -view of short-term t e s t s for mutagens stated that the advantage of t h i s test was the capacity to sele c t an e f f e c t i v e , non-toxic concentration of the mutagen which was not subsequently d i l u t e d i n the t e s t . These properties make the test p a r t i c u l a r l y useful for measuring mutagens occurring at low concentrations. Green and Muriel (1976) also recognized the benefits of th i s test i n that i t can measure mutation frequency with l e v e l s of mutagens which are l i k e l y to be found i n the environment. The p r i n c i p l e of the fl u c t u a t i o n test has been used s u c c e s s f u l l y to detect the presence of muta-gens i n aqueous extracts of feces using TA 100 and TA 98 (Kuhnlein and Kuhnlein, 1980; Kuhnlein, et a l . , 1981). Summary The l i t e r a t u r e reviewed i n t h i s chapter indicates that f e c a l mutagenic a c t i v i t y i s r e l a t e d to r e l a t i v e r i s k f or colon cancer. Several studies have demonstrated that f e c a l mutagenicity can be detected and measured using sen-s i t i v e s t r a i n s of the b a c t e r i a ScttmomZta. typkmvJium. The f l u c t u a t i o n t e s t for mutagens uses t h i s b a c t e r i a , and i t has been employed s u c c e s s f u l l y i n the measurement of mutagenic a c t i v i t y i n human feces. Considerable support for r e l a t i o n s h i p s between di e t and f e c a l muta-g e n i c i t y , and between d i e t and r i s k f o r colon cancer also e x i s t s i n the l i t e r a t u r e . Diets high i n animal f a t and animal protein, and low i n vegetable protein and f i b e r have been associated with high r i s k for colon cancer and with high l e v e l s of f e c a l mutagenic a c t i v i t y . Conversely, di e t s high i n f i b e r and low i n animal protein and animal f a t have been associated with low r i s k for colon cancer and with low l e v e l s of f e c a l mutagenicity. Therefore, the present study was developed to examine the e f f e c t s of short-term consumption of hypothesized high- r i s k and low-risk di e t s for colon cancer on f e c a l mutagenic a c t i v i t y . 31 CHAPTER III METHODS Research Purpose and Plan An experimental research design was developed to test the hypothesis that short-term consumption of d i e t s considered to be h i g h - r i s k for colon cancer r e s u l t s i n mutagenic a c t i v i t y of feces at l e v e l s s i g n i f i c a n t l y greater than when low-risk d i e t s are consumed. Various theories have been proposed to explain the r e l a t i o n s h i p between di e t and r i s k for colon cancer. Examin-atio n of some parameters of feces and the e f f e c t s of short-term dietary changes on these was undertaken i n an attempt to elucidate those factors which might influence changes i n mutagenic a c t i v i t y and hence help to explain the e t i o l o g y of colon cancer. The development of d i e t s considered to be h i g h - r i s k or low-risk for colon cancer constitutes the f i r s t part of t h i s study. In r e l a t i o n to the habitual d i e t s of the s i x subjects who p a r t i c i p a t e d i n the study, low-risk and h i g h - r i s k d i e t s were devised. Fiber, meat and animal f a t are the dietary components thought to be r e l a t e d to r e l a t i v e r i s k for colon cancer. The low-risk d i e t s f o r t h i s study were lacto-ovo vegetarian and high i n f i b e r , while the hi g h - r i s k d i e t s were high i n meat and beef, and low i n f i b e r . The subjects consumed, i n sequence, baseline d i e t s representative of t h e i r habitual d i e t s for one week, low-risk d i e t s for two weeks and high r i s k d i e t s f o r two weeks. Fecal samples were c o l l e c t e d at the end of each of the diet periods. Aqueous extracts of the f e c a l samples were assayed to determine i f short-term dietary changes were rel a t e d to changes i n f e c a l mutagenicity. The samples were also examined for percent dry weight and pH to e s t a b l i s h i f these parameters were rel a t e d to d i e t or to mutagenic a c t i v i t y of the feces. L i f e s t y l e Questionnaire and Dietary Data C o l l e c t i o n Instruments L i f e s t y l e Questionnaire A questionnaire was developed to gather information on the subjects, some c h a r a c t e r i s t i c s of t h e i r normal d i e t s with respect to consumption of the f a t of meat and use of spices, recent dietary changes, use of medications, family and personal h i s t o r y of disorders of the g a s t r o i n t e s t i n a l t r a c t or cancer, bowel habits, smoking habits and use of alcohol. The questionnaire i s included as Appendix A. The information from the questionnaire was used to determine whether any d i e t a r y or l i f e s t y l e p r actices were followed which might influence the findings of the study. Food Frequency Questionnaire The food frequency questionnaire which appears i n Appendix B was developed to e l i c i t information on the frequency of consumption of foods which are sources of f i b e r , animal f a t , vegetable f a t , animal protein and vegetable protein. Consumption of these food components has been assoc-iated with r e l a t i v e r i s k f o r colon cancer. Subjects were required to e s t i -mate t h e i r consumption of each food item, and to check i n the appropriate column how often the designated portion of food was consumed. There were 10 possible choices of frequency of consumption, ranging from 'more than once d a i l y ' to 'never'. The information obtained through analysis of the food frequency questionnaire was used to characterize the normal di e t s of the subjects, and to e s t a b l i s h that n u t r i t i o n a l l y adequate, omnivorous d i e t s were h a b i t u a l l y consumed. It was also used to ensure that the base-l i n e d i e t s selected by the subjects for consumption during the f i r s t week of t h i s study were s i m i l a r to t h e i r h a b i t u a l d i e t s . D a i l y Record Forms Food record forms including the names of foods consumed, amounts consumed, methods of preparation, comments, recipes and explanations were used by the subjects to o u t l i n e a t y p i c a l days' intake to be consumed d a i l y during the f i r s t week of the study. The same form was used by the subjects to record t h e i r d a i l y food intakes throughout the f i v e weeks of the study. Space to record d a i l y use of medications and d a i l y bowel habits was added to the form f o r use throughout the study. The food record form i s shown i n Appendix C. Description of Subjects Six subjects, 5 female and 1 male, p a r t i c i p a t e d v o l u n t a r i l y i n t h i s study. They provided written consent by signing the consent form which appears i n Appendix D, as required by the U.B.C. Committee for Research Involving Human Subjects. The subjects ranged i n age from 25 to 33, and a l l had resided i n Canada for a minimum of 18 years. The l i f e s t y l e quest-ionnaire was administered p r i o r to the study to e l i c i t information regarding d i e t , bowel habits, use of medications, c i g a r e t t e s and alcohol, and personal and family medical h i s t o r y . A l l subjects were i n good health at the time of the study, and a l l reported normal bowel habits. Two subjects r e s p e c t i v e l y ingested the prescribed medications, Synthyroid and o r a l contraceptives. One subject smoked an average of 9 cigarettes d a i l y throughout the study. A l l subjects reported the occasional use of alcohol. Four of the s i x sub-j e c t s reported a deliberate increase i n t h e i r consumption of foods high i n f i b e r content and a decrease i n the consumption of meat, e s p e c i a l l y red meat, over the previous f i v e years. The information obtained regarding l i f e s t y l e was not subsequently r e l a t e d to the findings of t h i s study since the number of subjects was too small for s i g n i f i c a n t findings to be gener-ated. Information and Instructions Provided to Subjects Dietary Guidelines and Information S p e c i f i c guidelines f o r the vegetarian and high meat d i e t s were developed based on the ou t l i n e s f o r the baseline d i e t s which were provided by the subjects. Individual guidelines for the numbers of d a i l y servings of f r u i t s and vegetables, whole grain cereal products, j u i c e , vegetable o i l and margarine, eggs, milk products and meat were provided to each subject as shown i n Appendix E. These foods were considered to be s i g n i f i c a n t sources of f i b e r , animal protein, animal f a t , vegetable protein and vegetable f a t - the food components thought to be associated with r i s k for colon cancer. Since a l l of the subjects were knowledgeable i n n u t r i t i o n , recom-mended ranges f o r d a i l y consumption of these nutrients were included i n the guidelines. The basis for the i n d i v i d u a l guidelines was also provided f o r the subjects' information. Appendix F shows general d e s c r i p t i v e guidelines for the vegetarian and high meat d i e t s , including i d e n t i f i c a t i o n of the s a l i e n t components of the d i e t s . B r i e f descriptions of the three diets to be consumed throughout the f i v e weeks of the study were also included. This information was pro-vided to the subjects. In addition, each subject was provided, i n advance, with a time schedule for the study, including the dates f o r each di e t period and for the f e c a l c o l l e c t i o n s . The time schedule appears i n Appendix G, Instructions f o r C o l l e c t i o n of Fecal Samples, Each subject received i n s t r u c t i o n s for the c o l l e c t i o n of f e c a l samples, including the timing of the c o l l e c t i o n s and the handling of the sample containers. Appendix H shows the i n s t r u c t i o n s provided. 35 Development of the Experimental Diets The duration of the study period was 5 weeks, during which a l l of the subjects consumed i n sequence a t y p i c a l baseline d i e t for one week, a l a c t o -ovo vegetarian d i e t for two weeks, and a di e t high i n beef and other red meats and low i n f i b e r for the f i n a l two weeks. Each subject was required i n i t i a l l y to provide on the d a i l y food record form a d e t a i l e d o u t l i n e of a t y p i c a l day's food and beverage intake which could be consumed d a i l y on days 1 to 7 of the study. The nutrient composition: of t h i s baseline d i e t plan was analyzed with respect to crude f i b e r , animal protein, vegetable protein, animal f a t , vegetable f a t and food servings of f r u i t s and vegetables, whole grain products, eggs and milk pro-ducts. This analysis was used as the basis from which guidelines f o r d a i l y food consumption on the vegetarian and high meat d i e t s were developed for each subject. The i n d i v i d u a l guidelines were designed to make the vegetarian and high meat d i e t s as d i f f e r e n t as possible from each other, and to provide eating patterns hypothesized to be at low-, and h i g h - r i s k f o r colon cancer i n r e l a t i o n to each subjects' baseline d i e t . For the vegetarian d i e t , maximum amounts of animal f a t , vegetable f a t and animal protein, minimum amounts of vegetable protein, and a l i m i t e d range for f i b e r consumption formed the basis for the guidelines. Maximum amounts of f i b e r , vegetable f a t and vegetable protein and minimum amounts of animal f a t and animal protein formed the basis for the guidelines for the high meat d i e t . The guidelines f o r a l l of the' subjects had a s i m i l a r b a s i s , as shown i n Appendix E. Description of the Experimental Diets Baseline Diet Subjects consumed t h e i r previously selected t y p i c a l day's intake on each day of the f i r s t week of the study. They were permitted to substitute s p e c i f i c food items within the same food group, provided that a s i m i l a r preparation method was used. No a d d i t i o n a l items could be included a f t e r the f i r s t day on the baseline d i e t , nor could foods be deleted. Two subjects made changes on the f i r s t day of the study, and the i n d i v i d u a l guidelines f o r the subsequent d i e t s were revised accordingly. Lacto-ovo Vegetarian Diet During weeks 2 and 3, subjects were asked to r e f r a i n from consuming meat, f i s h or poultry, to consume foods high i n f i b e r and foods low i n animal f a t , to l i m i t consumption of vegetable o i l and margarine, to use low-fat dairy products, to u t i l i z e legumes, nuts and seeds to replace animal protein sources, and to adhere to the i n d i v i d u a l guidelines provided. Subjects were free to plan t h e i r meals within the framework provided. High Meat Diet During weeks 4 and 5, subjects were asked to consume at least 3 ounces of cooked,, lean beef and an a d d i t i o n a l 3 ounces of any lean red meat d a i l y . The f a t from the meat could be consumed i n addition, i f desired. They were asked to s e l e c t foods low i n f i b e r and high i n animal f a t , to consume only refine d cereal products, and to l i m i t t h e i r consumption of f r u i t s , vegetables and j u i c e s as indicated i n the i n d i v i d u a l guidelines. The subjects were again free to plan t h e i r meals within the framework provided. C o l l e c t i o n of Dietary Data and Fecal Samples C o l l e c t i o n of Dietary Data Daily food records, as shown i n Appendix C, were maintained by the subjects throughout the f i v e weeks of the study. Coding of dietary inform-ation for computer analysis was done d i r e c t l y from these food records, as was manual analysis of food servings. The number of d a i l y bowel movements and the d a i l y use of cig a r e t t e s or medications were also recorded on t h i s form. C o l l e c t i o n of Fecal Samples Subjects made 24-hour f e c a l c o l l e c t i o n s on days 6 and 7 of the base-l i n e d i e t , and 3 consecutive 24-hour c o l l e c t i o n s beginning on the 11th days of the vegetarian and high meat d i e t s r e s p e c t i v e l y . The procedure of f e c a l sampling thus provided the maximum time for changes i n f e c a l mutagenicity and f e c a l c h a r a c t e r i s t i c s to occur. Four c o l l e c t i o n days were designated on the vegetarian and high meat die t s to allow f o r subjects who might not have a bowel movement each day. However, consecutive c o l l e c t i o n s were required f o r the analysis of wet and dry weights. A t o t a l of 8 f e c a l c o l l e c t i o n s were made by each subject. The f e c a l samples were c o l l e c t e d d i r e c t l y into new, l a b e l l e d 1 L p l a s t i c containers (Lab-Tek 4725). A separate container was used f o r c o l l e c t i n g each sample i n the event that more than one sample was provided i n a 24-hour c o l l e c t i o n period. Subjects were instructed to avoid con-taminating the f e c a l samples with urine or menstrual blood. The f e c a l samples were immediately frozen by placing the containers i n a styrofoam box of dry i c e . Samples were removed a f t e r each di e t period, and stored at -20° C. The subjects were permitted to proceed with the next di e t as soon as f e c a l c o l l e c t i o n s f o r one d i e t were completed. Assays for Mutagenic A c t i v i t y of Feces The f l u c t u a t i o n test of L u r i a and Delbruck 0-943). as modified by Green and Muriel (1976), Green et a l , (1976) and Kuhnlein et a l . (1981) was used to assay for mutagenic a c t i v i t y of aqueous extracts of f e c a l samples. The test was selected for i t s capacity to detect mutagens at very low, non-toxi c concentrations such as might be found i n the environment ( H o l l s t e i n et a l . , 1979). The two h i s t i d l n e auxotrophic d e r i v a t i v e s of SalxnonoXZci typhsimuAAUm, TA 98 and TA 100, were used as tester s t r a i n s i n the f l u c t u -ation t e s t . Mutagenic a c t i v i t y was assessed by measuring the reversion from a h i s t i d i n e requiring to a h i s t i d i n e prototrophic phenotype. The reversion i n TA 98 i s mainly induced by frameshift mutations, whereas TA 100 i s s e n s i t i v e to base-pair substitutions i n DNA. The use of two tester s t r a i n s allowed detection of changes i n a spectrum of f e c a l mutagens as a r e s u l t of dietary changes, as well as changes i n the o v e r a l l mutagenicity of the feces. Preliminary Assays for Mutagenic A c t i v i t y A preliminary ser i e s of experiments was performed to assess the capacity of bovine serum, human urine and aqueous extracts of human feces to trap the known carcinogens methyl nitro-nitroso-guadinine and 4-nitroquino-line-N-oxide. The r e s u l t s of these experiments are reported i n Appendix I. The test for mutagenicity used i n these studies was the standard "Ames-test" for mutagenicity. In respect to f e c a l mutagenicity, i t became evident during these experiments that aqueous f e c a l extracts were toxic and i n t e r f e r e d with the mutagenicity assay. It was therefore decided to use the f l u c t u a t i o n test of Green and Muriel (1976) for the assessment of f e c a l mutagenicity. This assay i s more s e n s i t i v e , and can be c a r r i e d out at lower extract concentrat-ions. Fluctuation Test Media and solution s. The following media and solutions were used during the f l u c t u a t i o n t e s t s . Davis-Mingioli s a l t s 1 JL d i s t i l l e d , deiohizedwwater 2 g KH 2P0 4 7 g K 2HP0 4 1 g •CNH,t)2S0I| 0.25 g trisodium c i t r a t e 0.1 g MgSO^HjjO 5 mg bromcresol purple Davis medium for overnight culture Davis medium for f l u c t u a t i o n experiment Nutrient broth 100 ml Davis-Mingioli s a l t s s o l u t i o n 2 ml 20% D-glucose 0.4 ml D-biotin (0.1 mg/ml) 1.25 ml L - h i s t i d i n e (1 mg/ml) 120 ml Davis-Mingioli s a l t s s o l u t i o n 2.4 ml 20% D-glucose 0.48 ml D-biotin (0.1 mg/ml) 30 u l L - h i s t i d i n e (1 mg/ml) 1 L d i s t i l l e d , deionized water 8 g Difco Bacto nutrient broth 5 g NaCl Bacteria. The bac t e r i a tester s t r a i n s used were ScUmomLtd typhmuAMm s t r a i n s TA 98 and TA 100. TA 98 i s s e n s i t i v e to mutagens causing frameshift mutations i n DNA, and TA 100 i s s e n s i t i v e to mutagens causing base-pair sub-s t i t u t i o n s . Preparation of f e c a l extracts. Each 24-hour f e c a l c o l l e c t i o n was thawed, weighed, and homogenized with an equal weight of d i s t i l l e d water. Aliquots o were centrifuged at 4 C for 50 minutes at 150,000 g. The supernatants were s t e r i l i z e d by consecutive f i l t r a t i o n through f i l t e r s of pore s i z e 0.45yum and 0.2 ^ m res p e c t i v e l y . The f e c a l extracts were stored i n 5 ml tubes at -20° C. Test procedure. Series of subcultures were prepared of ba c t e r i a SalmoneL&CL typkXmixfihm s t r a i n s TA 98 and TA 100. Ten ml aliquots of nutrient broth were inoculated under s t e r i l e conditions from a master culture of ba c t e r i a , and the cultures were grown overnight at 37° C with aeration to a concentration of 10 9/ml. Dimethyl sulfoxide to a f i n a l concentration of 10% was added to the cultures, and 1 ml aliquots were stored i n l i q u i d nitrogen. The same batch of subcultures was used throughout the experiments. On the evening preceding each seri e s of experiments, an overnight b a c t e r i a l culture was prepared by inoculating 10 ml Davis overnight culture medium with 1 ml b a c t e r i a l subculture, and growing overnight with aeration at 37° C to a concentration of 10 9/ml. The culture was held on i c e u n t i l needed. One ml or l e s s of f e c a l extract was added to a s o l u t i o n containing 120 ml Davls-Mingioli s a l t s , 2.4 ml 20% D-glucose, 0.48 ml D-biotin, 30 y l D - h i s t i d i n e and 1.2 ml b a c t e r i a TA 98 or TA 100 overnight culture d i l u t e d to 10 5/ml. Two ml portions of t h i s s o l u t i o n were dispensed into each of 50 test o tubes, covered with aluminum f o i l , and incubated at 37 C for 5 days i n a walk-in incubator. The l i m i t e d amount of h i s t i d i n e present i n the culture medium l i m i t s the growth of the auxotrophic b a c t e r i a to about 5*10 8/ml. Bacteria which have reverted to a prototrophic phenotype, however, can grow to a high density. Tubes containing revertants can e a s i l y be scored since the high concentration of b a c t e r i a a c i d i f i e s the medium and induces a color change of the bromcresol purple of the medium from purple to yellow. A c o n t r o l s o l u t i o n without f e c a l extract was tested with each sample to e s t a b l i s h the spontaneous mutation rate of the b a c t e r i a under the con-d i t i o n s of the experiment. An increase i n the number of revertant tubes when extract was present indicated the presence of a mutagen. In addition, one sample of f e c a l extract at concentration 0.25 yl/ml was tested on a sequence of experimental days to confirm the s t a b i l i t y of the mutagens present i n the f e c a l extract during storage at -20° C as well as the r e l i a b -i l i t y of the experiments. The r e s u l t s appear i n Appendix J. The f e c a l mutagens were found to be stable over time when stored at -20° C, and the r e s u l t s proved to be reproducible a f t e r storage under these conditions. Determinations of Fecal Sample C h a r a c t e r i s t i c s Wet weight, dry weight, percent dry weight and pH were determined for c o r r e l a t i o n with mutagenicity data. The t o t a l wet weights of the f e c a l samples were i n i t i a l l y determined and recorded. To c a l c u l a t e the t o t a l dry weights and percent dry weights of the f e c a l samples, 10 to 20 g portions of f e c a l homogenate were a i r dried f o r three days at 45 C and reweighed. The pH of each homogenate was measured against a standard buffer s o l u t i o n pH 7.00, using an Orion pH meter model 601A, and an Orion combination electrode No. 91-05. Dietary Analysis Food and nutrient analyses of the food frequency questionnaires and the d a i l y food records from the three d i e t periods were performed to estab-l i s h the following: - c h a r a c t e r i s t i c s of the subjects' habitual d i e t s - s i m i l a r i t i e s between the subjects' habitual d i e t s and the baseline d i e t s which they selected -compliance with the guidelines on each of the d i e t s - v a l i d i t y of assuming the vegetarian d i e t as 'low-risk' and the high meat d i e t as 'high-risk' f o r the purposes of t h i s study. Nutrient analyses of the food frequency questionnaires and the d a i l y food records were performed at the Computer Center of the Uni v e r s i t y of B r i t i s h Columbia using the General Nutrient Analysis program of the School of Home Economics. Data from the food frequency questionnaires were coded i n weekly intakes, and data from the d a i l y food records were coded d i r e c t l y on a d a i l y basis. The food frequency nutrient analysis was subsequently adjusted to give mean d a i l y r e s u l t s . The nutrient intake means and standard devia-tions were calculated for each subject and for the group f o r each of the three d i e t s . The analysis included energy i n k i l o c a l o r i e s , t o t a l protein, t o t a l f a t , carbohydrate, crude f i b e r , calcium, i r o n , vitamin A, thiamine, r i b o f l a v i n , n i a c i n and vitamin C. Total dietary f i b e r was also estimated using the food tables i n Composition of Foods (Paul and Southgate, 1978). Components were added to the computer program to allow the a d d i t i o n a l analysis of animal protein, vegetable protein, animal f a t and vegetable f a t 42 since analysis of d a i l y consumption of these nutrients was e s s e n t i a l i n evaluating the r e l a t i v e r i s k of the d i e t s f or colon cancer. The d a i l y food records were also used to determine the number of servings consumed on each d i e t of meat, legumes, nuts and seeds, milk pro-ducts, eggs, f r u i t s and vegetables, j u i c e , c e real products and f a t s and o i l s . This information was required to evaluate compliance with the dietary guide-l i n e s i n terms of food servings, and the r e l a t i v e r i s k of the d i e t s f or colon cancer. Analysis of Fecal Mutagenicity The r e s u l t s of the f l u c t u a t i o n t e s t s f or mutagenic a c t i v i t y of f e c a l samples obtained on the three d i e t s were analyzed to e s t a b l i s h whether changes i n mutagenicity did i n fa c t occur as a r e s u l t of short-term dietary changes. The average o v e r a l l mutation frequency per tube of the f l u c t u a t i o n test on each d i e t at each extract concentration was calculated by assuming a Poisson d i s t r i b u t i o n of mutation events among the tubes. Dose-response curves were plotted for each of the d i e t s using each of the b a c t e r i a l t e s t e r s t r a i n s , and the curves were integrated and corrected for the mutation frequency i n the absence of f e c a l extract. The c o r r e c t i o n was based on the number of revertant tubes at extract concentration 0.0yl/ml for a l l tests done on the same experiment day. Percent change i n mutagenicity obtained with the high meat di e t over the baseline and vegetarian d i e t s was calculated. S t a t i s t i c a l Analyses S t a t i s t i c a l analyses of the parameters of f e c a l samples examined i n t h i s study were performed to e s t a b l i s h the s i g n i f i c a n c e of changes i n f e c a l mutagenic a c t i v i t y over the three d i e t periods, and to attempt to explain the mechanism which might be involved i n the changes observed. Non-parametric s t a t i s t i c a l tests were chosen since the dose-response to the f e c a l extracts i n the f l u c t u a t i o n tests was not l i n e a r . S t a t i s t i c a l Analysis of Fecal Mutagenicity The changes i n mutagenicity over the three d i e t s were tested f o r s i g n i f i c a n c e using the chi-square test ( S i e g e l , 1956). The t o t a l number of tubes containing revertants and the t o t a l number containing no revertants"at each extract concentration for each di e t were used f o r t h i s t e s t . The sign-test for two rel a t e d samples (Siegel, 1956) was used to test the s i g n i f i c a n c e of the d i r e c t i o n of change i n mutagenicity among the d i e t s . This test uses plus and minus signs to denote the d i r e c t i o n of di f f e r e n c e between two observations, and tests the s i g n i f i c a n c e of the number of times each sign occurs. The sign-test was also used to e s t a b l i s h whether the o v e r a l l changes i n mutagenicity were consistent f o r a l l s i x subjects. The t o t a l number of revertant tubes over a l l concentrations of f e c a l extract, corrected f o r the number of revertant tubes i n the absence of extract, was averaged over each die t period f o r each subject. Corresponding values f o r each subject were considered as matched pai r s since considerable v a r i a t i o n i n the l e v e l s of mutagenicity among the subjects was apparent. Analysis of Parameters of Fecal Samples The Kruskal-Wallis one-way analysis of variance by ranks (Siegel, 1956) was used to test f o r differences i n wet weight, dry weight, percent dry weight and pH of f e c a l c o l l e c t i o n s as w e l l as the number of d a i l y bowel movements among die t s and among subjects. In the Kruskal-Wallis a n a l y s i s , each of the observations i s replaced by a number denoting the rank of the observation In the s e r i e s . The test determines whether a s i g n i f i c a n t d i f f e r e n c e e x i s t s between the sums of ranks of the samples being compared. The Kruskal-wallis analysis was performed among die t s to examine the e f f e c t s of dietary changes on the f e c a l parameters. S i g n i f i c a n t differences among subjects could indicate that i n d i v i d u a l genetic influence may a f f e c t the f e c a l parameters studied. In addition, the sign-test was used to test the s i g n i f i c a n c e of an observed decrease i n the number of d a i l y bowel move-ments when the subjects changed from the high meat di e t to the vegetarian d i e t . Correlations Among Fecal Mutagenicity and Other Fecal Parameters The Spearman rank cor r e l a t i o n s c o e f f i c i e n t (Siegel, 1956) measures the extent of agreement between the ranking of observations for two variables i n the same sample. This test was used to examine associations among the various parameters of the f e c a l samples. Fecal mutagenicity, wet weight, dry weight, percent dry weight and pH were considered. This t e s t was also used to test f o r c o r r e l a t i o n of the r e s u l t s of the f l u c t u a t i o n tests using TA 98 and TA 100, to determine whether the same spectrum of f e c a l mutagens was being measured on each of the d i e t s . CHAPTER IV RESULTS AND DISCUSSION. Analysis of Diets In t h i s study, s i x subjects consumed the following d i e t s i n sequence: a baseline d i e t of the subjects' choice for one week, a "low-risk" vegetarian diet for two weeks, and a "high-risk" high meat di e t for two weeks. A food frequency questionnaire was taken on each subject before the study began to define usual patterns of food and nutrient intake. Analysis of Food Frequency Questionnaires The mean nutrient composition of the subjects' habitual d i e t s derived from the food frequency questionnaires i s shown i n Table 1. A l l of the sub-j e c t s generally reported the consumption of omnivorous di e t s comprised" of a wide v a r i e t y of food items. The d i e t s complied with recommendations for a n u t r i t i o n a l l y adequate di e t (Health and Welfare Canada, 1975) with one exception. The mean reported intake of i r o n was marginally below the Recom-mended Daily Nutrient Intake for Canada, and subjects #2 and #3 reported d a i l y intakes of i r o n below the recommended 14 mg. T o t a l protein exceeded the Recommended D a i l y Nutrient Intake by about 50%, with 71% of the protein from animal o r i g i n . Consumption of high l e v e l s of animal protein has been associated with h i g h - r i s k for colon cancer. However, the use of dairy products was foremost among those foods containing animal protein, with the food items most frequently consumed being milk and cheese. Animal fat of dairy o r i g i n has been shown i n epidemiological studies not to c o r r e l a t e with incidence of colon cancer (Reddy et a l . , 1980b). The consumption of beef, which has been associated strongly with colon cancer (Maier et a l . , 1974; Reddy et a l . , 1974; Hentges et a l . , 1977) was reported only a maximum of twice weekly by any subject i n the food frequency TABLE 1 Mean Dai l y Nutrient Intakes Derived from Food Frequency Questionnaires and Baseline Diets Subj ect 1 Subject 2 Subj ect 3 Subj ect 1 4 . •  FF 1 B 2 FF B FF B FF B Energy Ckcals) 1,789 1,567 1,696 1,291 1,381 1,532 1,589 1,458 T o t a l protein (g) 82 83 -69 53 69 61 78 62 Animal protein (g) 52 57 50 39 54 46 46 41 Vegetable protein (g) 28 21 16 8 11 11 29 18 T o t a l f a t (g) 70 54 74 52 54 79 55 61 Animal f a t (g) 36 23 47 41 44 49 35 33 Vegetable f a t (g) 31 17 20 2 6 19 17 23 Carbohydrate (g) 218 202 188 155 159 154 208 167 Crude f i b e r (g) 10 6 8 7 7 5 10 5 Dietary f i b e r (g) 38 22 27 18 22 18 36 20 Calcium (mg) 791 935 924 894 1,082 930 915 571 Vitamin C (mg) 232 149 203 138 126 101 255 145 Vitamin A (I.U.). 12,636 7,018 8,676 7,893 11,531 9,165 13,730 4,890 Thiamine (mg) 1.26 3.21 1. 19 0.83 0. 77 0.92 1. 28 1. 08 R i b o f l a v i n (mg) 1.85 . 1.03 1,83 1. 28 1. 95 1.68 2. 16 1. 18 Ni a c i n (mg) 21. 81 10.65 13. 56 13. 36 16. 52 - 13,45 19. 40 14. 43 Iron (mg) 14. 59 16.09 11.59 8.56 9. 84 8.23 14. 42 10. 20 ^ood frequency 2 B a s e l i n e d i e t TABLE 1 (continued) Subj ect 5 Subject 6 Group Means FF 1 B 2 FF B FF S.D. B S.D. Energy (kcals) 1,848 2,097 1,920 1,737 1,704 196 1,601 362 Tot a l p r o t e i n (g) 94 98 81 87 79 9 73 21 Animal protein (g) 60 67 51 67 52 5 52 17 Vegetable protein (g) 28 30 27 20 23 8 18 7 To t a l f a t (g) 83 104 86 96 70 14 73 28 Animal f a t (g) 51 67 50 58 44 7 45 23 Vegetable f a t (g) 23 33 26 29 21 8 20 13 Carbohydrate (g) 190 196 213 136 196 22 168 36 Crude f i b e r (g) 7 6 9 4 9 2 6 2 Dietary f i b e r (g) 30 26 34 23 31 6 21 5 Calcium (mg) 847 537 824 700 897 104 760 355 Vitamin C (mg) 149 109 245 175 202 53 136 49 Vitamin A (I.U.) 12,065 11,229 7,779 24,976 11,070 2,336 10,627 9,241 Thiamine (mg) 1. 42 1.47 1. 51 1. 42 1.24 0.26 1. 46 0. 86 Ri b o f l a v i n (mg) 2. 44 1.92 2. 22 2. 64 2.08 0.24 1. 41 3. 27 Niacin (mg) 19. 54 25.95 18. 04 19. 51 18.15 2.90 16. 10 6. 40 Iron (mg) 15. 74 16.67 15. 50 14.05 13.61 2.22 12. 15 4. 16 Food frequency 2 B a s e l i n e diet 48 questionnaires. Vitamin C intakes as determined from the food frequency question-naires exceeded the Recommended Dai l y Intakes by about 350%. This nutrient i s thought to act as an anticarcinogen i n the etiology of colon cancer by i n h i b i t i n g nitrosamine formation i n the gut (upton, 1980). The reported intakes of Vitamin A exceeded the recommendation by about 100%. This nutrient has not been proved to influence r i s k f o r colon cancer. The analysis f o r energy from the food frequency questionnaires i s l i k e l y l e s s accurate than analyses f o r other n u t r i e n t s . Sugar and h i g h l y r e f i n e d and processed sources of sugar were not included on the food frequency questionnaire since i t was designed to e l i c i t information about the consumption of protein, f a t and f i b e r . Therefore, the energy analysis i s incomplete. Analysis of the food frequency questionnaires revealed that the habitual d i e t s of a l l of the subjects tended to be high i n dietary f i b e r , ingested i n the form of f r u i t s , vegetables and whole g r a i n cereal products. Dietary f a t was moderate, as was consumption of meat and beef. Therefore, the habitual d i e t s of the subjects tended to be i n l i n e with the hypothesized low-risk d i e t f or colon cancer i n these respects. Comparison of Habitual and Baseline Diets Although)the baseline d i e t s were not expected to exactly r e f l e c t the food intakes recorded i n the food frequency questionnaires, Table 1 shows a general s i m i l a r i t y i n nutrient composition between the food frequency analyses and the baseline diets selected by each subject. The subjects' usual d i e t s , as shown by the food frequency questionnaires and the baseline d i e t s , tended to include high f i b e r foods such as whole grain products, vegetables and f r e s h f r u i t s . 49 Compliance with Guidelines for Experimental Diets Tables 2 to 7 show, for each subject, the estimated intakes on the baseline d i e t , the guidelines for the vegetarian and high meat d i e t s , and the actual intakes on a l l three d i e t s i n terms of food servings and f i b e r , fat and protein. The subjects did not comply completely with the guidelines for the vegetarian and high meat d i e t s . I t appeared that the requested servings of vegetables, f r u i t s and refined cereal products were not always palatable r e l a t i v e to the subjects' usual d i e t s . The subjects generally chose to consume poultry during the baseline d i e t i n preference to other types of meat. Since the guidelines for each subject were established from the baseline plan, and since the protein con-tent of poultry i s somewhat higher than that of red meats, i t was unreal-i s t i c for the subjects to double t h e i r intakes of animal protein using beef, lamb, pork and organ meats during the high meat di e t period. However, the guidelines were more c l e a r l y met i n terms of quantity of meat, and a l l subjects consumed at le a s t as much meat as was required during the high meat d i e t . There was a tendency on the part of the subjects to use eggs and cheese i n preference to legumes as substitutes for meat on the vegetarian d i e t . In f a c t , Appendix K shows that only two subjects had mean d a i l y i n -takes of legumes i n excess of H cup. The use of eggs and cheese resulted i n higher intakes of animal f a t and animal protein than requested i n the guidelines f o r the vegetarian d i e t . Only subject #3 achieved the desired intake of vegetable protein on the vegetarian d i e t . On the high meat d i e t , a l l subjects except subject #4 exceeded the guidelines for intakes of f r u i t and vegetables;- Only three subjects com-p l e t e l y avoided whole grain products, and only subjects #5 and #6 completely eliminated reduced f a t milk products on the high meat d i e t . A l l of the subjects except #6 consumed more j u i c e on the high meat d i e t than on the TABLE 2 Expected and Actual Mean Intakes of Selected Foods and Nutrients Subject Number 1 Baseline diet Vegetarian diet High meat diet Foods Estimate 1 Actual Mean Range Guideline2 Actual Mean Range Guideline Actual Mean Range Beef, 1 oz. 1 0.0 0.0 0.0 *3.0 1».3 3.0-8.0 \ 3.0 - (-3.0)3 Other meat, f i s h , poultry, 1 oz. J k.O 0.0 0.0 - 2.2 0.0-3.0 Whole milk products, 1 cup or equiv. 0.5 0.9 0.0-2.0 0.0 0.3 0.0-1.2 2.0 0.5 0.0-1.0 Reduced f a t milk products, 1 cup or equiv. 1.0 0.7 0.5-1.1 0.2 1.3 0.0-2.5 0.0 0.7 0.0-1.0 Egg, 1 0.0 0.0 - 0.0-1.0 0.1 0.0-1.3 As desired 0.1 0.0-0.3 Vegetable o i l or margarine, 1 tsp. 3.0 0.5 0.0-1.0 <12.0 2.0 0.0-5.0 <12.0 2.7 0.0-5.0 F r u i t , vegetable, legumes, seeds, 1/2 cup 5.0 It.6-6.7 7.0 8.0 1*.1-12.7 3.0 3.3 1.0-5.5 Juice, 1/2 cup 1.5 • 1.5 - As desired 0.7 0.0-2.0 6.0 2.3 0.6-8.0 Whole grain cereal, 1 exchange 5.0 l+.l 3.5-1*.5 6.0 5.2 3.5-7.5 0.0 0.0 -Refined c e r e a l , 1 exchange 0.0 0.0 - 0.0 0.3 0.0-1.0 As desired It.8 3.0-9.0 Selected nutrients Estimate 1 Mean ±S.D. Guideline 2 Mean ±S.D. Guideline 2 Mean +S.D. Crude f i b e r (g) 8 6 1 11-22 15 6 0-U 5 3 Animal f a t (g) 16 23 11 0-8 9 13 >32 33 15 Vegetable fat (g) 17 17 2 17-56 30 15 17-56 22 15 Animal protein (g) l*lt 57 18 0-22 21 20 >88 1*7 15 Vegetable protein (g) 20 21 1 >1*0 38 Ik 0-20 21 6 1 T y p i c a l day's intake preplanned by subject 2Prcvided by experimenter, based on subject's estimated baseline diet 3Subject free to select meat, f i s h , poultry or additional beef TABLE 3 Expected and Actual Mean Intakes of Selected Foods and Nutrients Subject Number 2 Baseline diet Vegetarian diet High meat diet Foods Estimate 1 Actual Mean Range Guideline2 Actual Mean Range Guideline2 Actual Mean Range Beef, 1 oz. | 3.0 0.7 0.0-!*.0 0.0 0.0 - ±3.0 5.5 3.0-7.5 Other meat, f i s h , poultry, 1 oz. J 1.0 o.o-i+.o 0.0 0.0 - (>3.0)3 1.0 0.0-2.0 Whole milk products, 1 cup or equiv. 2.5 2.8 2.0-3.0 0.0 1.7 o.5-h.o 3.0 2.1 0.5-3.5 Reduced fat milk products, 1 cup or equiv 0.5 0.5 0.1-1.0 3.0 0.6 0.0-1.0 0.0 0.3 0.0-0.5 Egg, 1 0.0 0.0 - 0.0-1.0 0.1 0.0-1.5 As desired 0.0 -Vegetable o i l or margarine, 1 tsp. 0.0 0.0 - <12.0 2.3 0.0-6.0 <12.0 h.l 0.0-12.1 F r u i t , vegetable, legumes, seeds, 1/2 cup h.O it.7 1.5-6.0 5.0 6.0 3.5-9.0 3.0 3.0 1.0-5.0 Juice, 1/2 cup 0.0 0.0 - As desired 0.0 - <6.0 0.5 0.0-1.0 Whole grain c e r e a l , 1 exchange 3.0 3.0 2.0-lt.O 1*.0 h.l 3.0-5.0 0.0 0.2 0.0-2.0 Refined c e r e a l , 1 exchange 0.0 0.9 0.0-3.0 , 0.0 0.3 0.0-1.5 As desired 3.9 1.5-6.0 Selected nutrients Estimate 1 Mean ±S.D. Guideline 2 Mean ±S.D. Guideline 2 Mean ±S.D. Crude f i b e r (g) 6 7 2 8-17 10 5 0-3 1* 2 Animal f a t (g) 36 hi 9 0-18 2k 10 >72 75 18 Vegetable fat (g) 3 2 2 3-56 17 18 3-56 17 23 Animal protein (g) hi 39 6 0-2h 27 11 >9h 62 13 Vegetable protein (g) 12 8 3 >23 19 l l * 0-12 15 5 1 T y p i c a l day's intake preplanned by 2Provided by experimenter, based on subject subject's estimated baseline diet 3Subject free to select meat, f i s h , poultry or additi o n a l beef 0TABLE 1* Expected and Actual Mean Intakes of Selected Foods and Nutrients Subject Number 3 Baseline diet Vegetarian diet High meat diet Foods Estimate 1 Actual Mean Range Guideline2 Actual Mean Range Guideline2 Actual Mean Range Beef, 1 oz. 1 n 1.3 0.0-5.0 0.0 0.0 - 13.0 6.1 3.0-8.0 Other meat, f i s h , poultry, 1 oz. J 1.3 0.0-3.0 0.0 0.0 - (>3.0)3 1.0 0.0-5.0 Whole milk products, 1 cup or equiv. 2.0 , 2.7 1.5-3.5 0.0 1.9 1.0-3.5 1*.0 1.7 0.5-3.8 Reduced fat milk products, 1 cup or equiv. 0.0 0.1* 0.2-0.5 1+.0 0.7 0.2-1.0 0.0 0.2 0.0-0.5 Egg, 1 0.0 0.6 0.1-2.0 0.0-1.0 0.1* 0.0-2.0 As desired <0.1 0.0-0.5 Vegetable o i l or margarine, 1 tsp. 3.0 3.5 2.0-6.0 <12.0 2.3 0.0-7.0 <12.0 2.1* 0.0-9.0 F r u i t , vegetable, legumes, seeds, 1/2 cup h.O l * . l 2.5-5.5 5.0 h.9 2.7-6.3 3.0 3.3 1.5-6.0 Juice, 1/2 cup 0.5 0.7 0.5-1.0 As desired 0.5 0.0-1.0 <6.0 1.1 0.0-3.5 Whole grain c e r e a l , 1 exchange 2.0 1.1 1.0-1.8 lt.0 U.5 2.5-8.0 0.0 0.2 0.0-1.0 Refined c e r e a l , 1 exchange 1.0 1.3 0.5-3.0 0.0 1.0 0.0-3.0 As desired 5.3 2.5-8.0 Selected nutrients Estimate 1 Mean ±S.D. Guideline 2 Mean ±S.D. Guideline 2 Mean ±S.D. Crude f i b e r (g) 1* 5 3 6-12 9 5 0-2 5 2 Animal f a t (g) 1+1 1*9 18 0-20 29 10 >80 70 29 Vegetable fat (g) 6 19 7 • 6-56 25 15 • 6-56 25 15 Animal protein (g) 1+5 1*6 13 0-23 31* 10 >90 55 12 Vegetable protein (g) 6 11 2 <12 25 13 0-6 18 6 1 Typical day's intake preplanned by subject 2Provided by experimenter, based on subject's estimated baseline diet 3SubJect free to select meat, : f i s h , poultry or additional beef TABLE 5 Expected and Actual Mean Intakes of Selected Foods and Nutrients Subject Number k Baseline diet Vegetarian diet High meat diet Foods Estimate 1 Actual Mean Range Guideline2 Actual Mean Range Guideline2 Actual Mean Range Beef, 1 oz. 1 1.6 0.0-U.O 0.0 0.0 - ±3.0 5.7 3.0-7.0 Other meat, f i s h , poultry, 1 oz. J . 3-5 2.1 0.0-h.O 0.0 0.0 - (>3.0) 3 0.7 0.0-3.0 Whole milk products, 1 cup or equiv. 2.0 1.1 1.0-1.5 0.0 1.1 0.0-2.0 3.0 0.5 0.0-2.0 Reduced fat milk products, 1 cup or equiv. 0.5 0.5 0.5-0.7 3.0 1.0 0.3-2.0 0.0 0.8 0.2-1.3 Egg, 1 0.0 0.0 - 0.0-1.0 0.8 0.0-2.0 As desired 0.6 0.0-2.0 Vegetable o i l or margarine, 1 tsp. k.Q 1.0-5.5 <12.0 3.2 1.0-7.0 <12.0 2.3 0.0-6.0 F r u i t , vegetable, legumes, seeds, 1/2 cup 7.0 6.1* 5.0-8.0 8.0 7.0 1* .1-9.7 lt.0 3.1* 2.0-l*.0 Juice, 1/2 cup 0.5 0.5 - As desired 1.2 0.0-2.0 <6.0 1.0 -Whole grain c e r e a l , 1 exchange 2.5 2.5 2.0^3.5 lt.0 1+.2 0.0-6.0 0.0 0.2 0.0-2.0 Refined c e r e a l , 1 exchange 0.0 0.3 0.0-2.0 0.0 0.1 0.0-1.0 As desired 1*.0 2.0-5.0 Selected nutrients Estimate 1 Mean ±S.D. Guideline 2 Mean ±S.D. Guideline 2 Mean . ±S.D. Crude f i b e r (g) 8 6 1 12-21* 10 1* 0-1* 3 1 Animal fat (g) 38 33 20 0-19 29 21* >75 60 19 Vegetable f a t (g) 26 23 7 25-56 37 27 26-56 12 8 Animal protein (g) 1*2 1*1 8 0-21 33 15 >81* 61* 15 Vegetable p r o t e i n (g) 20 18 3 >1*0 32 17 0-20 19 6 1 Typical day's intake preplanned by subject 2Provided by experimenter, based on subject's estimated baseline diet 3SubJect free to select meat, f i s h , poultry or additional beef TABLE 6 Expected and Actual Mean Intakes of Selected Foods and Nutrients Subject Number 5 Baseline diet Vegetarian diet High meat diet Foods Estimate 1 Actual Mean Range Guideline2 Actual . Mean Range Guideline2 Actual Mean Range Beef, 1 oz. I 6.0 2.0 0.0-6.0 0.0 0.0 - 23.0 5.7 3.0-11.0 Other meat, f i s h , poultry, 1 oz. J 5.8 3.0-9.0 0.0 0.0 - U3.0 ) 3 3.1 0.0-6.0 Whole milk products, 1 cup or equiv. 0.5 0.2 0.0-1.0 0.0 0.7 0.0-1.5 2.0 0.6 0.0-3.0 Reduced fat milk products, 1 cup or equiv. 0.5 0.2 0.0-0.8 2.0 0.9 0.0-2.0 0.0 <0.1 0.0-0.5 Egg, 1 2.0 2.0 - 0.0-1.0 0.7 0.0-2.0 As desired 1.6 0.0-2.0 Vegetable o i l or margarine, 1 tsp. 9.0 7.8 5.0-10.0 <12.0 5.5 0.0-15.0 <12.0 . It.9 0.0-11.0 F r u i t , vegetable, legumes, seeds, 1/2 cup lt.0 3.8 3.0-lt.5 7.0 7.7 2.5-12.5 lt.0 5.3 2.5-8.0 Juice, 1/2 cup 1.0 1.0 - As desired 0.2 0.0-1.0 <6.0 6.It 0.0-1.5 Whole grain c e r e a l , 1 exchange 5.0 6.3 5.0-7.0 lt.0 5.2 3.0-6.5 . 0.0 0.0 -Refined c e r e a l , 1 exchange 0.0 0.8 0.0-lt.O 0.0 0.3 0.0-3.0 As desired 5.8 2.0-8.3 Selected nutrients Estimate 1 Mean ±S.D. Guideline 2 Mean ±S.D. Guideline 2 Mean ±S.D. Crude f i b e r (g) 7 6 2 9-20 12 6 0-3 5 2 Animal f a t (g) 55 67 29 0-28 22 19 >110 76 1+6 Vegetable fat (g) 28 33 10 28-56 1+0 2lt 28-56 26 11 Animal protein (g) 59 67 13 0-30 2lt 17 >118 71 21+ Vegetable protein (g) 27 29 5 >5lt It It 26 0-27 20 6 Typical day's intake preplanned by subject 3Subject free to select meat, f i s h , poultry or additional beef Provided by experimenter, based on subject's estimated baseline diet TABLE 7 Expected and Actual Mean Intakes of Selected Foods and Nutrients Subject Number 6 Baseline diet Vegetarian diet High meat diet Foods Estimate 1 Actual Guideline 2 Actual Guideline 2 Actual Mean Range Mean Range Mean Range Beef, 1 oz. [ £ n 1.8 0.0-7.0 0.0 0.0 - *3.0 U.8 3.0-8.0 Other meat, f i s h , poultry, 1 oz. J o. u It.8 2.0-8.0 0.0 0.0 (>3.0)3 2.5 O.O-lt.O Whole milk products, 1 cup or equiv. 0.5 0.0 - 0.0 0.6 0.0-3.0 3.0 1.0 0.0-3.0 Reduced f a t milk products, 1 cup or equiv 1.0 1.3 1.0-1.5 3.0 2.2 1.0-3.0 0.0 0.0 -Egg, 1 1.5 1.8 1.0-3.0 0.0-1.0 0.9 0.0-2.0 As desired 0.8 0.0-2.0 Vegetable o i l or margarine, 1 tsp. 6.0 7.3 it.0-10.0 <i2.0 5.3 0.0-15.0 <12.0 3.1 0.0-6.0 F r u i t , vegetable, legumes, seeds, 1/2 cup 6.0 6.2 5.0-7.5 7.0 8.0 3.0-12.0 lt.0 It.6 3.0-6.0 Juice, 1/2 cup 1.0 1.0 - As desired 0.7 0.0-1.5 <6.0 0.3 0.0-1.5 Whole grain c e r e a l , 1 exchange 2.0 2.3 1.0-3.0 lt.0 It.3 0.0-7.5 0.0 0.0 -Refined c e r e a l , 1 exchange 0.0 0.3 0.0-1.5 0.0 0.3 0.0-3.0 As desired It.3 1.0-6.8 Selected nutrients Estimate 1 Mean ±S.D. Guideline 2 Mean ±S.D. Guideline 2 Mean ±S.D. Crude f i b e r (g) 5 It 0.1 8-15 9 It 0-3 It 2 Animal fat (g) 5h 58 22 0-27 35 26 >108 56 19 Vegetable f a t (g) 23 29 17 23-56 29 23-56 19 7 Animal protein (g) 58 67 18 0-29 38 26 >ll6 56 25 Vegetable protein (g) 19 20 3 >38 36 16 0-19 17 5 1 Typical day's intake preplanned by subject 3Subject free to select meat, f i s h , poultry or additi o n a l beef 2Provided by experimenter,. based on subject's estimated baseline diet 56 vegetarian d i e t . The estimated intakes of vegetable f a t and o i l provided by the subjects for the baseline d i e t were very low. Only two subjects exceeded 2 tablespoons d a i l y . The intakes remained co n s i s t e n t l y less than 2 tablespoons on the vegetarian and high meat d i e t s . Animal f a t consumption on the base-l i n e d i e t was higher than estimated i n advance by the subjects, except for subject #4, and only subject #5 did not exceed the guidelines for animal f a t during the vegetarian d i e t . This r e l a t e s to the high consumption of dairy products on the vegetarian d i e t , and to the f a c t that no subject completely replaced whole milk products with low f a t milk products. Despite the fact that the subjects did not always meet the prescribed guidelines of the d i e t s , Tables 2 to 7 c l e a r l y e s t a b l i s h differences i n com-pos i t i o n between the d i e t s . A l l s i x of the subjects consumed les s f i b e r , more animal f a t and more animal protein on the high meat d i e t than on the vegetarian d i e t . Composition of Baseline, Vegetarian and High Meat Diets The mean number of d a i l y food servings from various food groups and the range of the mean numbers of servings over a l l of the s i x subjects are shown i n Table 8. The consumption of whole milk products and eggs was un-changed between the vegetarian and high meat d i e t s . A change i n the approp-r i a t e d i r e c t i o n was observed for a l l other food groups, except j u i c e , when subjects moved from the vegetarian d i e t to the high meat d i e t . The number of food servings consumed d a i l y by i n d i v i d u a l subjects on a l l of the d i e t s appears i n Appendix K. Tables 9 to 14 show the mean d a i l y nutrient intakes for each subject on each of the three d i e t s . Table 15 shows the group mean d a i l y intakes of protein, f a t and f i b e r on each of the three d i e t s . These food components are thought to be re l a t e d to the r e l a t i v e r i s k for colon cancer. Intakes of t o t a l TABLE 8 Mean D a i l y Number and Range of Food Servings i n Baseline, Vegetarian and High Meat Diets for A l l Subjects Food group Baseline d i e t Vegetarian d i e t High meat di e t Mean Range Mean Range Mean Range Beef - 1 oz. 1.3 0.0-2.0 0.0 5.4 4.3-6.1 Other meat - 1 oz. 3.4 1,0-5.8 0.0 1.7 0.7-3.1 Legumes - cup Q.Q 0.7 0.2-1,4 0.Q 0.0-0.1 Nuts - 1 tbsp. 0.3 0.Q-2.Q 1.7 0.2-3.2 0.5 0.0-1.2 Whole milk products - 1 cup 1.2 0.0-2.8 1.1 0.3-1.9 1.1 0.5-2.1 . Low f a t milk products - 1 cup 0.6 0.4-1.3 1.1 0.6-2.2 0.3 Q.0-0.8 Egg - 1 0.7 Q.0-2.0 0,5 0.1-0.9. 0.5 0.0-1.6 Animal f a t - 1 tsp, l . o 0.0-3.2 0.7 0,0-1,4 2.3 1,1-4.6 Vegetable f a t - 1 tsp. 4.Q 0.0-7.8 3,4 2.0-5.5 3.2 2.3-4.9 Fruit/Vegetables - % cup 5.0 3.8-6.4 6.3 4.2-7.4 3-7 3.0-4.5 Juice - H cup 0,9 0,0-1.5 0.4 0,0-1.2 1.0 0.3-2.3 Whole grain cereals 1 exch. 3.1 1.1-6,3 4,6 4,1-5.2 0.1 0.0-0.2 Refined cereals - 1 exch. Q.6 0,0-1.3 0.4 Q.1-1.0 4,7 3.9-5.8 TABLE 9 Mean Dai l y Nutrients i n Baseline, Vegetarian and High Meat Diets, Subject Number 1 Baseline d i e t Vegetarian diet High meat di e t Nutrient _ _ Mean ±S.D. Mean ±S.D. Mean ±S.D. Energy (kcals) 1,567 263 1,839 393 1,802 400 To t a l p r o t e i n (g) 83 18 63 . 23 74 15 Animal p r o t e i n (g) 57 18 21 20 47 15 Vegetable p r o t e i n (g) 21 1 38 14 21 6 Combination p r o t e i n (g) 4 1 5 2 4 6 To t a l f a t (g) 54 12 46 22 61 27 Animal f a t (g) 23 11 9 13 33 15 Vegetable f a t (g) 17 2 30 15 22 15 Combination f a t (g) 14 2 7 5 5 9 Carbohydrate (g) 202 27 299 83 205 42 Crude f i b e r (g)_ 6 1 15 6 5 3 Dietary f i b e r (g) 22 3 42 13 20 11 Calcium (mg) 935 631 913 499 617 304 Vitamin C (mg) 149 30 117 66 117 74 Vitamin A (I.U.) 7,018 3,367 14,558 8,960 34,239 38,085 Thiamine (mg) 3. 21 Q.20 1.77 0.99 1. 26 0.24 R i b o f l a v i n (mg) 1. 03 0.89 1.76 0.39 3. 68 3.06 Nia c i n (mg) 10. 65 1.9.6 17.64 3.41 27. 49 11.25 Iron (mg) 16. 09 0.32 16.42 6.54 20. 98 9.21 00 TABLE 10 Mean Daily Nutrients i n Baseline, Vegetarian and High Meat Diets, Subject Number 2 Baseline d i e t Vegetarian diet High meat di e t Nutrient 6  Mean ±S.D. Mean ±S.D. Mean ±S.D. Energy (kcals) 1,291 199 1,597 254 2,349 616 T o t a l p r o t e i n (g) 53 6 55 ; 1 7 85 16 Animal p r o t e i n (g) 39 6 27 11 62 13 Vegetable p r o t e i n (g) 8 3 19 14 15 5 Combination protein (g) 6 2 9 4 9 8 To t a l f a t (g) 52 9 59 22 111 35 Animal f a t (g) 41 9 24 10 75 18 Vegetable f a t (g) 2 2 17 18 17 23 Combination f a t (g) 10 5 18 9 16 17 Carbohydrate (g) 155 52 221 40 263 76 Crude f i b e r (g) 7 2 10 5 4 2 Dietary f i b e r (g) 18 5 29 8 15 4 Calcium (mg) 894 143 1,197 380 857 224 Vitamin C (mg) 138 57 150 72 113 52 Vitamin A (I.U.) 7,893 3,659 11,962 9,546 7,821 13,432 Thiamine (mg) 0. 83 0.37 0. 90 0.23 0. 95 0.23 R i b o f l a v i n (mg) 1. 28 0.26 1. 56 0.57 1. 44 0.38 Niacin (mg) 13. 36 2.50 12. 82 3.98 16. 67 3.15 Iron (mg) 8. 56 1.22 10.73 2.20 13. 56 3.39 TABLE 11 Mean D a i l y Nutrients i n Baseline, Vegetarian and High Meat Diets, Subject Number 3 Baseline d i e t Vegetarian diet High meat diet Mean ±S.D. Mean ±S.D. Mean ±S.D. Energy (kcals) 1,532 178 1,648 304 2,203 367 To t a l p r o t e i n (g) 61 12 ' 65 84 15 Animal protein (g) 46 13 34 10 55 12 Vegetable p r o t e i n (g) 11 2 25 13 - 18 6 Combination protein Cg) 5 3 7 4 8 5 To t a l f a t Cg) 79 14 69 18 107 37 Animal f a t Cg) 49 18 29 10 70 29 Vegetable f a t Cg) 19, 7 25 15 25 15 Combination f a t Cg) 11 13 15 10 10 7 Carbohydrate Cg) 154 26 195 39 229 40 Crude f i b e r Cg) 5 3 10 5 • 5 2 Dietary f i b e r Cg) 19 6 28 9 16 5 Calcium (mg) 930 97 1,159 29.6 696 159 Vitamin C (mg) 101 40 76 42 100 . 70 Vitamin A (LIT.) 9,165 5,363 8,296 3,645 8,517 - 8,311 Thiamine (mg) 0. 92 0.34 0.91 0.19 . 1. 23 0.36 R i b o f l a v i n (mg) 1. 68 0.34 1.49 0.34 1. 72 0.55 Niacin (mg) 13. 45 3.46 15.90 5.70 22. 37 5.60 Iron (jag) 8. 23 1.81 10.94 2.78 16. 05 4.81 ON o TABLE 12 Mean Dal l y Nutrients i n Baseline, Vegetarian and High Meat Diets, Subject Number 4 Nutrient Baseline d i e t Vegetarian diet High meat di e t Mean ±S.D. Mean ±S.D. Mean ±S.D. Energy Cecals) 1,458 226 1,741 485 1,678 274 T o t a l p r o t e i n (g) 62 10 68 85 14 Animal protein (g) 41 8 33 15 64 15 Vegetable p r o t e i n (g) 18 3 32 17 19 6 Combination p r o t e i n (g) 3 7 26 4 3 5 T o t a l f a t (g) 61 19 72 34 80 24 Animal f a t A ( g ) 33 20 29 24 60 19 Vegetable f a t (g) 23 7 37 27 12 8 Combination f a t (g) 4 9 6 . 6 8 7 Carbohydrate (g) 117 28 22Q 43 153 36 Crude f i b e r (g) 6 1 10 4 3 1 Dietary f i b e r (g) 20 2 41 18 12 3 Calcium (mg) 571 117 1,100 313 786 319 Vitamin C (mg) 115 49 187 60 107 32 Vitamin A (I.U.) 4,890 2,837 8,684 9,282 19,867 22,404 Thiamine (mg) 1.08 0.24 1.57 1.35 0.99 0.24 R i b o f l a v i n (mg) 1.18 0.31 1.70 0.49 1.71 0.71 Niacin (mg) 14.43 3.58 13.23 j 4.05 17.63 3.97 Iron (mg) 10.20 1.68 12,68 4.98 11.78 1.81 TABLE 13 Mean D a i l y Nutrients i n Baseline, Vegetarian and High Meat Diets, Subject Number 5 . . . Baseline d i e t Vegetarian d i e t High meat di e t Nutrient ' Mean ±S.D. . Mean ±S.D, Mean + S.D. Energy (kcals) 2,098 387 2,098 474 '2,545 661 T o t a l p r o t e i n (g) 98 13 77 ; 23 105 23 Animal protein Cg) 67 13 24 17 71 24 Vegetable p r o t e i n (g) 2& 5 44 26 2Q 6 Combination p r o t e i n (g) 1 1 6 9 14 13 To t a l f a t Cg) 104 35 77 37 129 55 Animal f a t Cg) 67 29 22 19- 76 46 Vegetable f a t Cg) 33 10 40 24 26 11 Combination f a t Cg) 1 1 12 16 27 20 Carbohydrate Cg) 1 % " 11 277 56 223 59. Crude f i b e r Cg) 6 2 • 12 6 5 2 Dietary f i b e r Cg). 26 3 35 10 17 14 Calcium (mg) 537 498 9181 411 486 166 Vitamin C (mg) 1Q9 49 150. 89 1Q9 51 Vitamin A (I.U.). 11,229 9,147 18,546 12,300 13,908 16,89.0 Thiamine Cmg) 1. 47 0.27 1. 78 1.76 1. 79 0.57 R i b o f l a v i n (mg) 1. 92 0.11 2, 22 1.62 2. 49 1.64 Ni a c i n (mg) 25. 95 6.50 17, 57 6.22 28. 22 8.79. Iron (mg) 16. 67 3.28 16. 66 4.27 18. 72 4.22 ON TABLE 14 Mean D a l l y Nutrients i n Baseline, Vegetarian and High Meat Diets, Subject Number 6 Nutrient Baseline d i e t Vegetarian diet High meat die t Mean ±S.D. Mean ±S.D. Mean ±S.D. Energy (kcals) 1,737 349 2,176 568 1,945 368 To t a l protein (g) 87 19 79 27 85 16 Animal protein (g) 67 18 38 26 56 19 Vegetable protein (g) 20 3 36 16 17 5 Combination protein (g) 0 0 5 5 12 12 T o t a l f a t (g) 9.6 28 94 47 96 27 Animal f a t (g) 58 22 35 26 56 25 Vegetable f a t (g) 29 17 49 29. 19 9 Combination f a t (g) 9 10 8 11 21 15 Carbohydrate (g) 136 . 12 258 40 182 44 Crude f i b e r (g) 4 1 9 4 4 2 Dietary f i b e r (g) 23 4 31 8 14 3 Calcium (mg) 700 158 1,251 5Q3 522 224 Vitamin C (mg) 175 43 182 89 93 43 Vitamin A (I.U,) 24,976 12,658 18,736 9, 079 11,969 12 ,363 Thiamine (mg) 1.42 0.47 1,47 0.85 ' 1. 41 0.42 R i b o f l a v i n (mg) 2.64 1.41 2,12 0.77 2. 03 1,23 Niacin (mg) 19.51 5,77 13,11 3.89 20. 85 4.58 Iron (mg) 14.05 4.41 14,38 3.41 . . 14. 39 2.74 ON TABLE 15 Mean and Range of Daily Nutrient Intakes on Baseline, Vegetarian and High Meat Diets for A l l Subjects Baseline d i e t Vegetarian diet High meat diet Nutrient Mean ±S.D. Range Mean + S.D. Range Mean + S.D. Range Energy (kcals) 1601 362 975-2638 1852 It69 110lt-3ltllt 2092 51*8 1072-li388 Total protein (g) 73 21 lt2-119 68 22 32-122 86 19 1*8-157 Animal protein (g) 52 17 30-93 29 18 1-92 59 18 26-117 Vegetable protein (g) 18 7 lt-36 32 19 7-101 18 6 7-36 Combination protein (g) 3 1+ 0-19 6 5 0-32 8 9 0-ltl Total f a t (g) 73 28 36-llt9 69 31* 15-191 98 ltl 25-287 Animal fat (g) h5 23 llt-107 21* 20 0-101 62 30 11 -208 Vegetable fat (g) 20 13 0-50 33 2lt 0-113 20 15 0-82 Combination f a t (g) 8 9 0-33 11 11 0-ltlt 15 15 0-67 Carbohydrate (g) 168 36 67-250 2lt6 63 136-li30 210 61 9 2 - 3 9 8 Crude f i b e r (g) 6 2 3-9 11 5 3-27 • It 2 l-ft.3 Dietary f i b e r (g) 22 5 11-30 3k 12 15-71* 16 6 6-38 • Calcium (mg) 760 355 2l*lt-2213 1099 Itllt 300-2338 663 268 19I+-I280 Vitamin C (mg) 136 ^9 U5-21+2 lltlt 79 11-378 . 107 55 : 9-301 Vitamin A (I.U.) 10,627 921*1 1855-1*1,151 13,531 9867 Ilt22-lt0,275 15,856 22,058 1026-!t2,213 Thiamine (mg) 1.1*6 0.86 0.52-3.51 l . l t l 1.09 0.5lt-7.57 1.27 0.1+5 0.It 8-2.73 Riboflavin (mg) 1.1*1 3.27 0.89-7.07 1. 81 0.81t 0.92-7.07 2.16 •1.67- 0,75^10.16 Niacin (mg) 16.10 6.1*0 6.99-36.1*8 15. 03 l*.95 6.9lt-33.70 22.lit 7.9U 11.19-lt7.7lt Iron (mg) 12.15 It.16 6.37-21.90 13. 66 It.79 6.38-31*. 60 15.89 5.71 7.l8-ltlt.76 65 protein, animal protein, t o t a l f a t and animal f a t increased when the subjects transferred from the vegetarian d i e t to the high meat d i e t , and intakes of vegetable f a t , vegetable protein and f i b e r decreased. The percentage of k i l o c a l o r i e s derived from animal f a t , vegetable f a t , t o t a l f a t , animal protein, vegetable protein and t o t a l protein for each sub-j e c t and for the group i s shown i n Table 16. The expression of these nutr-ients as a percentage of k i l o c a l o r i e s serves to emphasize the differences i n composition between the vegetarian and high meat d i e t s . The diff e r e n c e s i n diet composition between the low-risk, vegetarian d i e t and the h i g h - r i s k , high meat diet are more marked than the differences between the baseline and high meat d i e t s when expressed as a percentage of k i l o c a l o r i e s . The comparison of experimental d i e t s shows that, for the purpose of t h i s study, the vegetarian and high meat die t s represent eating patterns considered to be r e l a t i v e l y low-risk and hig h - r i s k for colon cancer. Analysis of Fecal Samples In t h i s study, each of the subjects c o l l e c t e d 8 f e c a l samples: two 24-hour samples at the end of the baseline d i e t period, three at the end of the vegetarian d i e t period, and three at the end of the high meat di e t period. These samples were analyzed for mutagenic a c t i v i t y using Salmonella typkwvJiiim TA 98 and TA 100. The percent dry weight and pH were also determined. Analysis of Mutagenic A c t i v i t y of Fecal Samples The r e s u l t s of the f l u c t u a t i o n tests for mutagenic a c t i v i t y of in d i v i d u a l f e c a l samples provided by each subject appear i n Appendix L. Table 17 gives the cumulative t o t a l number of f l u c t u a t i o n test tubes con-ta i n i n g revertant b a c t e r i a for a l l subjects on each d i e t and for each concentration of extract tested. These data were used to ca l c u l a t e the TABLE 16 Composition of Diets Expressed as Percent of K i l o c a l o r i e s for Each Subject and the Group Nutrient Subject 1 Subject 2 Subject 3 Subject l* Subject 5 Subject 6 Subjects 1-6 B 1 V 2 H 3 B 1 V 2 H 3 B 1 V 2 H 3 B 1 V 2 H 3 B 1 V 2 H3' B 1 V 2 H 3 B 1 V 2 H 3 Animal fat 13.2 It.2 16.6 28.2 13.lt 28.6 28.9 15.7 28.lt 20.1| 15.0 32.3 28.5 9.it 26.9 30.2 11*.5 25.9 25.1 11.8 26.6 Vegetable f a t 9.7 lit.8 10.8 1.1 9.3 6.6 10.9 13.7 10.1 lit.3 19.0 6.1* lit.3 17.3 9.2 15.1 20.2 8^8 11.2 16.1 8.6 Total fat 30.9 22.5 30.6 36.0 33.3 1+2.5 U6.3 37-7 1*3.6 37.7 37.0 1*3.0 ltlt.5 33.0 1*5.6 1*9.8 38.7 1*1*.2 1*1.2 33.7 ltl . 9 Animal protein lit.6 It. 5 10.5 12.1 6.8 10.5 12.0 8.3 10.0 11.2 7.6 15.2 12.8 It.5 11.1 15.5 6.9 11.5 13,1 6.3 13.3 Vegetable protein 5.2 8.2 It.6 2.6 It.6 2.5 2.8 6.0 3.3 5.0 7.It it.5 5.6 8.3 3.2 1*.5 6.7 3.1* l * . l * 7.0 3.5 Total protein 21.0 13.7 16.5 16.5 13.8 Ht.5 16.0 15.9 15.2 17.1 15.6 20.3 18.7 l i t . 6 16,5 20.0 11*.6 17.5 18.2 11*.7 16.5 b a s e l i n e diet 2Vegetarian d i e t 3High meat diet TABLE 17 Sum of Tubes Containing Each Revertant i Extract Bac t e r i a for Concentration Each Diet Period at Extract concentration (UL/ml) Baseline d i e t Vegetarian < diet High meat di e t TA 98 TA 100 TA 98 TA 100 TA 98 TA 100 0.0 198 233 312 312 281 312 0.52 310 257 507 372 526 379 1.04 350 338 5 a 477 561 514 2.08 367 408 592 618 584 638 4.17 423 421 636 665 643 705 8.33 464 504 723 763 804 822 Baseline d i e t = 600 tubes at each concentration. Vegetarian and high meat di e t s = 900 tubes at each concentration. average number of mutation events per f l u c t u a t i o n test tube at each concen-t r a t i o n of extract and on each d i e t , assuming a Poisson d i s t r i b u t i o n of mutation events. The r e s u l t s appear i n Table 18. Graphical representation of these r e s u l t s appears i n Figure 4 as average dose-response curves for the three d i e t periods. Figure 4 shows that f e c a l mutagenic a c t i v i t y was s i m i l a r on the baseline and vegetarian d i e t s using b a c t e r i a t e s t e r s t r a i n s TA 100 and TA 98, and increased on the high meat d i e t , e s p e c i a l l y at higher concentrations of f e c a l extract. Integration of the dose-response curves, corrected for the mutation frequency i n the absence of extract, shows that the average mutagenicity obtained with the high meat d i e t was 43% higher than the baseline d i e t using tester s t r a i n TA 100, and 36% higher using TA 98. In comparison with the vegetarian d i e t , the average mutagenicity obtained with the high meat di e t was 28% higher using TA 98, and 27% higher using TA 100. The integrated curve values are given i n Table 19. The r e s u l t s of the chi-square t e s t f o r changes i n mutagenicity over the three d i e t s are shown i n Table 20, and the s i g n i f i c a n c e of the r e s u l t s i s shown i n Table 21. The mutagenicity l e v e l s obtained with the baseline di e t were not s i g n i f i c a n t l y d i f f e r e n t from those obtained with the vegetarian di e t using bacteria tester s t r a i n s TA 98 or TA 100. However, the high meat die t resulted i n mutagenicity l e v e l s s i g n i f i c a n t l y higher (p<0.05) than those observed on the baseline and vegetarian d i e t s . This was shown for f e c a l extract concentrations 4.17 ul/ml and 8.33 ul/ml using TA 100, and for 8.33 ul/ml using TA 98. The s i g n i f i c a n c e of the d i r e c t i o n of change i n mutagenic a c t i v i t y using the sign-test f o r matched pai r s appears i n Table 22. The high meat di e t resulted i n consistently higher l e v e l s of mutagenicity over the base-l i n e and vegetarian d i e t s (p<0.05) at a l l concentrations of f e c a l extract using TA 100 and TA 98. There was no consistent d i r e c t i o n of change i n TABLE 18 Average Number of Mutation Events per Fluctuation Tube on Each Diet at Each Extract Concentration Extract concentration Cyl /ml ) Baseline di e t Vegetarian diet High meat diet TA 98 TA 100 TA 98 TA 100 TA 98 TA 100 0.0 0.40 0.49 0.43 0.43 0.37 0.43 0.52 0.73 0.56 0.83 0.53 0.88 0.55 1.04 0.88 0.83 0.92 0.76 0.98 0.85 2.08 0.95 1.14 1.07 1.16 1.05 1.23 4.17 1.22 1.21 1.23 1.34 1.25 1.53 8.33 1.48 1.83 1.63 1.88 2.24 2.45 Calculated assuming a Poisson d i s t r i b u t i o n of mutation events. 2.5 L i » • • • 1 • 2 4 6 8 2 4 6 8 Extract concentration ( u l / m l ) • Baseline d i e t A Vegetarian d i e t O High meat di e t FIGURE se-response Curves for Mutation Events Concentration of Extract on Baseline, 4 per F l u c t u a t i o n Test Tube at Each Vegetarian and High Meat Diets 71 TABLE 19 Integrated Values f or Mutation Events per Fluctuation Tube Expressed as a Dose-response Curve Diet TA 98 TA 100 Baseline 6.22 6.45 Vegetarian 6.59 7.31 High meat 8.44 9.28 Corrected f o r mutation frequency i n the absence of f e c a l extract. 72 TABLE 20 Results of Chi-square Analyses at D i f f e r e n t Levels of Extract Diet Tester Extract concentration (ul/ml) comparison s t r a i n 0 0.52 1.04 2.08 4.17 8.33 Baseline TA 100 2.52 0.27 0.00 0.05 0.00 0.11 vs. Vegetarian TA 98 0.37 2.98 0.58 3.12 0.00 1.78 Baseline TA 100 2.52 0.05 0.06 1.29 12.40 18.17 vs. High meat TA 98 0.44 6.43 2.25 1.99 0.11 38.74 Vegetarian TA 100 0.00 0.08 2.91 0.95 4.65 17.77 vs. High meat TA 98 2.26 0.74 0.60 0.12 0.10 27.75 TABLE 21 Sig n i f i c a n c e of Chi-square Analyses at Di f f e r e n t Levels of Extract Diet comparison Tester s t r a i n Extract concentration (ul/ml) 0, 0.52, 1.04, 2.8 4.17 8.33 Baseline TA 100 n.s. n.s. n. s. vs. Vegetarian TA 98 n.s. n.s. n.s. Baseline TA 100 n.s. B<H B<H vs. High meat TA 98 n.s. n.s. B<H Vegetarian TA 100 n. s. V<H V<H vs. High meat TA 98 n.s. n.s. V<H Differences i n mutagenicity are indicated for p<0.05. TABLE 22 Analysis of the Diet-induced Change i n Mutagenicity at Each Extract Level and for Each Subject by the Sign-test for Matched Pairs Diet Extract l e v e l s Subjects comparison TA 100 TA 98 TA 100 TA 98 Baseline vs. Vegetarian n. s. n. s. n.s. n.s. Baseline vs. High meat B<H B<H n.s. n.s. Vegetarian vs. High meat V<H V<H V<H n.s. Average numbers of revertant tubes at the same concentration of extract were considered as matched pai r s i n the s i g n - t e s t . 75 mutagenicity l e v e l s between the baseline and vegetarian d i e t s . Table 23 shows the average sum of revertant tubes for each subj ect on each of the d i e t s . Increased mutagenicity was observed on the high meat die t over the vegetarian d i e t f or a l l subjects using TA 100, and for f i v e of the s i x subjects using TA 98. These data were subjected to the sign-test for a consistent trend among the subjects towards higher l e v e l s of mutageni-c i t y on the high meat d i e t , and the r e s u l t s are shown i n Table 22. The d i r e c t i o n of change i n mutagenicity was s i g n i f i c a n t at the p<0.05 l e v e l using TA 100. The analysis of mutagenic a c t i v i t y of the f e c a l samples c o l l e c t e d over the three d i e t periods shows that the high meat di e t resulted i n o v e r a l l f e c a l mutagenic a c t i v i t y higher than that obtained on e i t h e r the baseline or vegetarian d i e t s . The high meat d i e t was the d i e t hypothesized to increase f e c a l mutagenicity and, therefore, the p o t e n t i a l r i s k f o r colon cancer. This r e s u l t supports the findings of Kuhnlein et a l (1981), who showed that vege-tarians had s i g n i f i c a n t l y lower f e c a l mutagenic a c t i v i t y than consumers of meat-containing d i e t s . The lacto-ovo vegetarian d i e t s consumed by subjects i n t h i s study resulted i n f e c a l mutagenic a c t i v i t y not s i g n i f i c a n t l y d i f f -erent from that obtained when the subjects consumed t h e i r h a b i t u a l omnivorous die t s which were high i n dietary f i b e r and r e l a t i v e l y low i n beef and animal f a t . I t i s not known i f a s t r i c t e r vegetarian d i e t would r e s u l t i n reduced f e c a l mutagenic a c t i v i t y over the short-term. However, Kuhnlein et a l (1981) observed l e v e l s of mutagenic a c t i v i t y that were s i m i l a r i n the feces of groups of both lacto-ovo vegetarians and vegans. It has not been determined which s p e c i f i c f a c t ors i n the environment within the colon are a l t e r e d as a r e s u l t of short-term dietary changes and may be responsible for the observed changes i n f e c a l mutagenic a c t i v i t y . The composition of f e c a l f l o r a i s one f a c t o r which has not c o n s i s t e n t l y responded to changes i n the components of the d i e t over short-term studies 76 TABLE 23 Average Sum of Revertant Tubes for Each Subject and Diet Period ™ Subject Dxet Tester Average over period s t r a i n 1 2 3 4 5 6 a l l subjects Baseline Vegetarian High meat TA 100 39.0 70.8 63.5 87.8 101.0 30.8 65.5 TA 98 63.0 74.0 95.8 92.5 91.5 57.8 79.1 TA 100 76.2 49.3 84.5 91.1 70.7 67.9 73.3 TA 98 89.2 57.5 89.5 111.3 65.7 81.3 82.4 TA 100 83.8 66.7 104.0 102.7 73.3 88.5 86.5 TA 98 102.5 45.7 98.5 102.0 75.8 102.3 90.8 (Cummings et a l . , 1978; Maier et a l . , 1974; Hentges et a l . , 1977; Reddy et a l . , 1974) However, the metabolic a c t i v i t y of f e c a l f l o r a was observed to increase when a high meat d i e t was consumed for 4 weeks (Reddy et a l . , 1974). Changes i n f e c a l excretion of b i l e acids and s t e r o l s have also been observed (Reddy et a l . , 1974; Cummings et a l . , 1979; Reddy et a l . , 1980c; U l l r i c h et a l . , 1981). These factors may be involved i n the changes i n f e c a l mutageni-c i t y which were observed. Analysis of Fecal Sample C h a r a c t e r i s t i c s The wet weight, dry weight and percent dry weight of each of the f e c a l samples c o l l e c t e d by each subject over the three d i e t periods are shown i n Tables 24 and 25. These data were subjected to the Kruskal-Wallis one-way analysis of variance by ranks, and the r e s u l t s appear i n Table 26. Examination of the 48 f e c a l samples c o l l e c t e d over t h i s study with respect to wet weight, dry weight and percent dry weight showed that there were no s i g n i f i c a n t changes i n these parameters as a r e s u l t of dietary changes. Therefore, there was no evidence that the high meat, low f i b e r d i e t resulted i n more concentrated s t o o l s . However, there was s i g n i f i c a n t v a r i a t i o n among subjects at the p<0.01 or p<0.001 l e v e l s f or a l l of these v a r i a b l e s . This suggests i n d i v i d u a l genetic influence on these f e c a l para-meters. Previous studies by Eastwood et a l . (1973a), Eastwood et a l . (1973b), Fuchs et a l . (1976) and Baird et a l . (1977) showed increased f e c a l weight when food sources of dietary f i b e r were increased by up to 16 g, but a p a r a l l e l f i n d i n g was not observed i n t h i s study when dietary f i b e r was i n -creased on the vegetarian d i e t by an average of 18 g. A s l i g h t l y increased energy intake on the high meat d i e t may be the r e s u l t of an increase i n volume of food consumed, and may account for maintaining the wet weight of the feces during the high meat d i e t period. There may also be a contribu-t i o n by i n d i g e s t i b l e protein as was hypothesized by Saunders (1980). TABLE 24 Wet Weights i n Grams of 24-hour Fecal C o l l e c t i o n s Sample Subject 1 Subject 2 Subject 3 Subject 4 Subject 5 Subject 6 B l i 81 245 71 38 93 213 B2 176 74 92 50 175 159 V I 2 154 212 88 121 98 313 V2 154 118 74 51 98 210 V3 103 75 67 100 225 360 HI 3 174 52 111 30 117 148 H2 169 246 105 70 90 109 H3 173 105 69 59 135 35 1 Baseline d i e t 2 Vegetarian d i e t 3 High meat di e t TABLE 25 Dry Weights i n Grams and Percent Dry Weights of 24-hour Fecal C o l l e c t i o n s Sample Subject 1 Subject 2 Subject 3 Subject 4 Subject 5 Subject 6 B l 1 25? (30. 4) 5 111, (45. 3) 14, (19. 8) 16, (41.2) 29, (31.1) 34, (16.1) B2 46, (26. 1) 42, (56. 2) 19, (20. 7) 25, (50.0) 48, (27.4) 23, (14.4) V I 2 40, (26. 1) 52, (24. 4) 19, (21. 4) 52, (43.0) 18, (18.6) 53, (17.0) V2 39, (25. 4) 29, (24. 3) 20, (26. 9) 21, (40.8) 24, (24.5) 34, (16.2) V3 27, (26. 0) 15, (20. 4) 18, (26. 9) 37, (37.4) 65, (28.8) 73, (20.4) HI 3 51, (29. 5) 21, (40. 0) 31, (28. 0) 9, (30.2) 30, (25.9) 30, (20.0) H2 45, (26. 7) 62, (25. 0) 27, (25. 8) 26, (36.5) 24, (26.4) 23, (21.2) H3 46, (26. 4) 29, (27. 7) 17, (24. 8) 20, (34.3) 40, (29.3) 8, (21.3) Baseline d i e t Vegetarian d i e t 3 High meat d i e t Dry weight 5 Percent dry weight TABLE 26 Kruskal-Wallis One-Way Analysis of Variance by Ranks of Fecal Sample Variables Variable Diet comparison Subject comparison Wet weights of f e c a l c o l l e c t i o n s Dry weights of f e c a l c o l l e c t i o n s % Dry weights of f e c a l c o l l e c t i o n s pH of f e c a l c o l l e c t i o n s Number of bowel movements per day n. s. n. s. n. s. n.s. n. s. 0.00K.p<0.01 p<0.001 p<0.001 p<0.001 0.001<p<0.01 Table 27 shows the pH of the 24-hour f e c a l c o l l e c t i o n s , and Table 28 shows the pH of a standard buffer s o l u t i o n which was used as a con t r o l f o r electrode d r i f t during the pH determinations. Normal pH of feces varies between 6.0 and 8.0, with increased pH observed i n the stools of cancer patients (Macdonald et a l . , 1978). The pH values observed i n t h i s study were within the range 5.80 to 8.34. Although subject #6 was responsible f o r the three samples below pH 6.0, one such sample occurred on each of the three d i e t s . The data i n Table 27 were also subjected to the Kruskal-Wallis one-way analysis of variance by ranks, and the r e s u l t s shown i n Table 26 show a tendency f o r v a r i a b l e pH values among i n d i v i d u a l subjects, but consistent pH values throughout the three d i e t s . Kuhnlein and Kuhnlein (1980) also observed persistent v a r i a b i l i t y among i n d i v i d u a l subjects although s i m i l a r c o n t r o l l e d d i e t s were consumed over a period of at l e a s t 47 days. The average number of bowel movements recorded d a i l y by the subjects on each of the d i e t s i s shown i n Figure 5. The Kruskal-Wallis one-way analysis of variance by ranks (Table 26) shows that the frequency of bowel movements varied s i g n i f i c a n t l y among the subjects at the 0.001<p<0.01 l e v e l . This v a r i a t i o n among subjects overrode any systematic changes i n the f r e -quency of bowel movements among the d i e t s when the Kruskal-Wallis analysis was performed. However, examination of the d i r e c t i o n of change i n the number of d a i l y bowel movements between the high meat and vegetarian d i e t s using the sign-test showed that the subjects had s i g n i f i c a n t l y fewer bowel movements on the high meat d i e t at the p<0.05 l e v e l . Consideration of a l l of the f e c a l c h a r a c t e r i s t i c s shows that i n d i v i d u a l v a r i a t i o n was s i g n i f i c a n t among subjects, but only the number of d a i l y bowel movements was affected by dietary changes. This supports the conclusion that dietary changes over more extended time periods are required for the occurrence of measureable a l t e r a t i o n s i n the c h a r a c t e r i s t i c s of TABLE 27 pH of 24-hour Fe c a l C o l l e c t i o n s Sample Subject 1 Subject 2 Subject 3 Subject 4 Subj ect 5 Subject 6 B l 1 7.11 7.29 8.34 6.87 7.23 6.73 B2 6.89 7.12 6,89 6.87 7.35 5.95 V I 2 6.34 7.37 6.24 6.71 7.01 6.04 V2 6.48 7.37 6.16 6.84 6.84 5.92 V3 6.61 7.36 6.63 6.76 7.03 6.31 HI 3 7.14 7.37 7.61 6.89 7.45 6.25 H2 7.03 6.97 7.11 7.07 7.91 5.80 H3 7.13 7.43 7.82 7.19 7.48 6.19 Baseline d i e t Vegetarian d i e t High meat d i e t 83 TABLE 28 pH Measurement Taken of a Standard Buffer Solution, pH 7.00, Used as a Control for Electrode D r i f t pH Reading time 7.00 at s t a r t 7.00 a f t e r 8 samples 7.02 a f t e r 16 samples 7.03 a f t e r 24 samples 7.03 a f t e r 32 samples 7.05 a f t e r 40 samples 84 Baseline diet [::::::j Vegetarian diet J High meat diet FIGURE 5 Mean Number of Daily Bowel Movements on Baseline, Vegetarian and High Meat Diets feces, or that i n d i v i d u a l v a r i a t i o n may have a genetic component. Correlations Among Fecal Parameters Correlations Among Fecal C h a r a c t e r i s t i c s Table 29 shows the r e s u l t s of Spearman rank c o r r e l a t i o n s among wet weight, dry weight, percent dry weight, and pH. The only s i g n i f i c a n t cor-r e l a t i o n observed was between wet weight and dry weight, p<0.001. This i s not s u r p r i s i n g since dry weight i s a component of wet weight. Correlations of Fecal Sample C h a r a c t e r i s t i c s with Mutagenicity Of the Spearman rank c o r r e l a t i o n s shown i n Table 29, only one f e c a l c h a r a c t e r i s t i c showed s i g n i f i c a n t c o r r e l a t i o n with mutagenicity. On the high meat d i e t , pH was p o s i t i v e l y correlated with mutagenicity using b a c t e r i a s t r a i n TA 100, p<0.001. However, there was no c o r r e l a t i o n using TA 98, An asso c i a t i o n between pH and mutagenicity has not been defined. Chambers and Burleson (1982) found that ozone-treated 1, 2-dimethylhydrazine, a known colon carcinogen was inact i v a t e d at high pH. Protection against colon cancer at low pH was proposed by Malhotra (1977), who hypothesized that low pH prevents a l k a l i p r e c i p i t a t i o n of protective mucus from mucosal c e l l s . I t i s not known whether t h i s mechanism could be rel a t e d to f e c a l mutagenicity. There may be some r e l a t i o n s h i p between pH and only some s p e c i f i c f e c a l mutagens, since TA 98 and TA 100 gave d i f f e r e n t r e s u l t s i n the Spearman rank c o r r e l a t i o n analysis. C o r r e l a t i o n between Mutagenicity with Tester Strains TA 98 and TA 100 As shown i n Table 29, s i g n i f i c a n t p o s i t i v e c o r r e l a t i o n using the Spearman rank c o r r e l a t i o n c o e f f i c i e n t was found between the mutagenicity l e v e l s observed using b a c t e r i a tester s t r a i n s TA 98 and TA 100 for the baseline d i e t , p<0.01 and the vegetarian d i e t , p<0.05. The c o r r e l a t i o n was not s i g n i f i c a n t for the high meat d i e t . Since TA 98 and TA 100 are 86 TABLE 29 Spearman Rank Cor r e l a t i o n s for Fec a l Sample Vari a b l e s Variables associated r g p Mutagenicity using TA 98 and using TA 100 Baseline d i e t Vegetarian diet High meat die t Mutagenicity and wet weight - TA 98 - TA 100 Mutagenicity and dry weight - TA 98 - TA 100 Mutagenicity and % dry weight - TA 98 - TA 100 Mutagenicity and pH TA 98 - Baseline d i e t - Vegetarian d i e t - High meat d i e t TA 100 - Baseline d i e t - Vegetarian d i e t - High meat d i e t Wet weight and dry weight Wet weight and % dry weight Dry weight and % dry weight Wet weight and pH Dry weight and pH % dry weight and pH 0.7483 0.4803 0.1559 -0.1528 -0.1137 -0.1974 0.3082 -0.0428 -0.1659 0.1503 -0.1089 0.0170 -0.1294 0.3344 0.7606 0.8540 0.3478 0.1219 -0.10.79 0.0199 0.3025 p<0.01 p<0.05 n.s. n.s. n.s. n. s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. p<0.001 p<0.001 n.s. n.s. n.s. n.s. n.s. 87 se n s i t i v e to mutagens which cause genetic damage i n d i f f e r e n t ways (Ames et a l . , 1973; Ames et a l . , 1975), i t appears that the high meat d i e t affected not only the o v e r a l l mutagen l e v e l s , but also the s p e c i f i c mutagens involved. Summary The r e s u l t s of t h i s study show that the o v e r a l l mutagenic a c t i v i t y of feces s i g n i f i c a n t l y increased over a period of two weeks as a r e s u l t of the consumption of a d i e t high i n meat and low i n f i b e r , which i s considered to be a hi g h - r i s k diet f o r colon cancer. The o v e r a l l d i r e c t i o n of change towards higher mutagenicity on the high meat di e t was s i g n i f i c a n t at a l l concentrations of f e c a l extract using TA 98 and TA 100. Wet weight, dry weight, percent dry weight and pH of f e c a l samples varied s i g n i f i c a n t l y among the subjects, but no changes were observed as a r e s u l t of dietary modification. The number of d a i l y bowel movements also varied s i g n i f i c a n t l y among the subjects, but there was a s i g n i f i c a n t trend towards fewer bowel movements when the subjects changed from the vegetarian di e t to the high meat d i e t . There was p o s i t i v e c o r r e l a t i o n of pH with f e c a l mutagenicity on the high meat d i e t using bacteria s t r a i n TA 100. There was s i g n i f i c a n t p o s i t i v e c o r r e l a t i o n between the mutagenicity observed using b a c t e r i a s t r a i n TA 98 and using TA 100 on the baseline and vegetarian d i e t s , but not .on the high meat d i e t . These r e s u l t s i n d i c a t e that short-term dietary changes r e s u l t i n s i g n i f i c a n t changes i n f e c a l mutagenicity that are not explained by changes i n wet weight, dry weight, percent dry weight or pH of feces. There appeared to be a diet-induced change i n the spectrum of mutagens involved. CHAPTER'V SUMMARY, LIMITATIONS AND IMPLICATIONS Summary of the Study 'This study tests the hypothesis that short-term consumption of d i e t s considered to be hi g h - r i s k f o r colon cancer r e s u l t s i n s i g n i f i c a n t l y greater mutagenic a c t i v i t y of feces than does consumption of low-risk d i e t s . The dietary components which are thought to be s i g n i f i c a n t are meat, e s p e c i a l l y beef, f a t , e s p e c i a l l y animal f a t , and f i b e r . Three experimental d i e t s were compared f o r t h e i r e f f e c t s on f e c a l mutagenicity. Six subjects p a r t i c i p a t e d i n t h i s study. They consumed i n sequence a baseline d i e t f o r one week, which represented each i n d i v i d u a l s ' h a b i t u a l d i e t ; a lacto-ovo, high f i b e r , vegetarian d i e t ; and a d i e t high i n meat, beef, animal protein and animal f a t and low i n f i b e r . The vegetarian and high meat die t s were res p e c t i v e l y considered to be low-risk and h i g h - r i s k for colon cancer, and were each consumed over two week periods. Individual guidelines f o r the hypothesized low-risk and high-ri s k diets were provided to the subjects based on food component intakes c a l -culated from outlines f o r the baseline d i e t s which were provided i n advance by the subjects. Food and nutrient analyses of the subjects' habitual d i e t s , and of the d a i l y intakes which were recorded during the three experi-mental di e t s were performed. This confirmed that the vegetarian and high meat d i e t s indeed f i t the models hypothesized to be low-risk and hi g h - r i s k f o r colon cancer i n r e l a t i o n to the habitual d i e t s . Fecal samples were c o l l e c t e d at the end of each d i e t period, and the mutagenic a c t i v i t y of aqueous extracts of the samples was assayed using b a c t e r i a l t e s t e r s t r a i n s of Salmonella tifphxjnuAAxm TA 98 and TA 100, i n the f l u c t u a t i o n test for mutagens. Ad d i t i o n a l parameters of the f e c a l samples were also examined i n an attempt to explain observed changes i n f e c a l mutagenicity r e l a t e d to dietary changes, and to suggest d i r e c t i o n s for further research. Wet weight, dry weight, percent dry weight and pH of the f e c a l samples were determined. The number of d a i l y bowel movements on each d i e t was also studied. Non-parametric s t a t i s t i c a l analyses were used i n t h i s study since the dose-response obtained i n the f l u c t u a t i o n tests f or mutagenicity was not l i n e a r . Chi-square and sign-test analyses of the changes i n mutagenicity on the experimental d i e t s were performed. Diet r e l a t e d changes i n the wet weight, dry weight, percent dry weight and pH of the f e c a l samples, and the number of d a i l y bowel movements were tested for s i g n i f i c a n c e using the Kruskal-Wallis one-way analysis of variance by ranks. A sign-test analysis of changes i n the mean number of d a i l y bowel movements on the three d i e t s was also performed. Significance of associations among a l l of the f e c a l parameters were tested using the Spearman rank c o r r e l a t i o n c o e f f i c i e n t . The r e s u l t s obtained from t h i s study confirm the i n i t i a l hypothesis that short-term consumption of d i e t s considered to be low-risk and h i g h - r i s k for colon cancer r e s u l t i n differences i n the mutagenic a c t i v i t y of feces. The h i g h - r i s k d i e t which was high i n meat, beef and animal protein and f a t , and low i n f i b e r resulted i n f e c a l mutagenicity s i g n i f i c a n t l y higher than that observed when a lacto-ovo vegetarian, high f i b e r d i e t was consumed. Mutagenicity from the h i g h - r i s k diet was also s i g n i f i c a n t l y higher than when an omnivorous d i e t , which was high i n f i b e r and low i n f a t , was consumed. The supposed low- and h i g h - r i s k experimental d i e t s which were com-pared i n t h i s study d i f f e r e d i n composition i n those foods and nutrients which have been most c l o s e l y correlated to the etiology of colon cancer, namely, f i b e r , animal protein, vegetable protein, animal f a t and vegetable f a t (Arm-strong and D o l l , 1975; H i l l and Drasar, 1972; Wynder, 1975).. The h i g h - r i s k d i e t which resulted i n increased o v e r a l l f e c a l mutagenicity contained a d a i l y 90 average of at l e a s t 6 ounces of meat, of which at l e a s t 3 ounces was beef, 0.1 servings of whole grain cereal products, 4.7 servings of r e f i n e d cereal products, 3.7 servings of f r u i t s and vegetables, 59 g animal protein, 62 g animal f a t and 16 g dietary f i b e r . The low-risk d i e t was lacto-ovo veget-arian, and contained a d a i l y average of 4.6 servings of whole grain cereal products, 0.4 servings of refined c e r e a l products, 6.3 servings of vegetables and f r u i t s , 29 g animal protein from dairy products, 24 g animal f a t from dairy products, and 34 g dietary f i b e r . The l a t t e r d i e t resulted i n s i g n i f -i c a n t l y lower mutagenic a c t i v i t y of feces i n comparison with the high meat d i e t . Lack of s i g n i f i c a n t differences related to dietary changes i n the wet weights, dry weights, percent dry weights and pH of the f e c a l samples indicates that short-term dietary changes do not a f f e c t these parameters, and they probably were not involved i n the observed changes i n f e c a l muta-ge n i c i t y . However, s i g n i f i c a n t differences were observed i n a l l of these parameters among the i n d i v i d u a l subjects, and t h i s suggests a r o l e for genetic influence or long-term environmental factors i n the determination of these f e c a l c h a r a c t e r i s t i c s . A s i g n i f i c a n t trend towards fewer d a i l y bowel movements when subjects changed from the vegetarian d i e t to the high meat d i e t was observed. How-ever, an ass o c i a t i o n between frequency of bowel movements and f e c a l mutageni-c i t y was not demonstrated i n the study. The s i g n i f i c a n t p o s i t i v e c o r r e l a t i o n observed between pH and f e c a l mutagenic a c t i v i t y at the p<0.001 l e v e l suggests that low pH stools may be associated with low r i s k for colon cancer. However, the r e s u l t s of t h i s study indicate that t h i s factor i s not amenable to short-term changes as a r e s u l t of dietary modification. P o s i t i v e c o r r e l a t i o n observed i n the r e s u l t s of the assays for f e c a l mutagenicity between the b a c t e r i a l t e s t e r s t r a i n s TA 98 and TA 100 on the i baseline and vegetarian d i e t s , and the lack of c o r r e l a t i o n on the high meat di e t s suggest that dietary modification changes the spectrum of f e c a l mutagens as w e l l as the o v e r a l l mutagenic a c t i v i t y of the feces. Limitations of the Study It i s recognized that the small number of subjects who p a r t i c i p a t e d i n t h i s study d i c t a t e d the emphasis on analysis of changes i n the parameters f o r i n d i v i d u a l subjects instead of f o r the o v e r a l l group. The r e s u l t s may also have been influenced by the f a c t that the normal d i e t s of the subjects more c l o s e l y resembled the experimental low-risk d i e t s , e s p e c i a l l y i n terms of high f i b e r and moderate meat and f a t content. As w e l l , the low-risk d i e t s which were consumed contained more animal f a t and animal p r o t e i n than the guidelines requested, and only l i m i t e d use was made of legumes, nuts and seeds as alternate sources of protein. Use of a more extreme "low-risk" d i e t might have produced more dramatic r e s u l t s i n the analysis of mutagenicity between the baseline and vegetarian d i e t s and of wet weight, dry weight, percent dry weight and pH among the three d i e t s . Implications The r e s u l t s of t h i s study have implications i n the p o t e n t i a l fOr modifying i n d i v i d u a l r i s k f o r colon cancer as assessed by f e c a l mutagenic a c t i v i t y . While i t i s not possible to determine from the study whether s p e c i f i c foods or nutrients are responsible f o r increasing or decreasing the l e v e l s of mutagenic a c t i v i t y i n feces, the combined e f f e c t s of t o t a l dietary changes were apparent i n l e s s than two weeks. The changes i n f e c a l mutagenicity suggest that the p o s s i b i l i t y e x i s t s for rapid modification of the r i s k f o r colon cancer of the i n d i v i d u a l . I t appears that the dietary changes necessary to modify f e c a l mutagenic a c t i v i t y are i n the realm of t y p i c a l Canadian d i e t s . Omnivorous diet s which were high i n f i b e r and low i n f a t , but including a moderate amount of meat, resulted i n f e c a l mutagenic a c t i v i t y not s i g n i f i c a n t l y d i f f e r e n t from that observed when a lacto-ovo vegetarian d i e t was consumed. It would be i n t e r e s t i n g to investigate whether or not the same magnitude of change i n mutagenicity would occur i f subjects consumed the hypothesized low-risk diet following the h i g h - r i s k d i e t . Further, i t would be useful to study the e f f e c t s of dietary changes on f e c a l mutagenicity i n subjects whose habitual d i e t s were more i n l i n e with the hypothesized "high—risk" d i e t . The scope for further research i s enormous. Epidemiology has already provided a v a r i e t y of hypotheses for the et i o l o g y of colon cancer based on d i e t as the operative environmental f a c t o r . Individual v a r i a t i o n s observed i n t h i s study for every parameter measured, and i n other studies for f e c a l m i c r o f l o r a (Hentges, 1977), bowel t r a n s i t time (Eastwood et a l , 1973), and f e c a l mutagenicity (Kuhnlein and Kuhnlein, 1980) i n d i c a t e the need for further studies with paired observations for the same subject under various conditions of d i e t . Controlled studies are needed to i d e n t i f y the e f f e c t s of i n d i v i d u a l n utrients, food items, food groups and food combina-tions on f e c a l mutagenic a c t i v i t y . Studies are needed to e s t a b l i s h the s p e c i f i c nature of the changes which occur i n the colon and are r e f l e c t e d i n the feces as a r e s u l t of dietary changes, over both the short-term and the long-term. Fecal mutagens need to be i d e n t i f i e d with respect to t h e i r formulation and t h e i r o r i g i n s . It has been hypothesized that cancer i s a disease the occurrence of which may be decades removed from i t s causative o r i g i n s . Prospective studies w i l l help to determine i f a rapid response to dietary changes, such as was observed i n t h i s study, w i l l indeed reduce the long-term r i s k f o r colon cancer. It i s hoped that r e s u l t s from t h i s study w i l l provide a base for future research to i d e n t i f y the etiology of colon cancer, and to i n i t i a t e recommendations which could help i n d i v i d u a l s to decrease t h e i r r i s k f o r t h i s disease. BIBLIOGRAPHY Ames, B.M. The detection of chemical mutagens with en t e r i c b a c t e r i a . In Chemical Mutagens: P r i n c i p l e s and Methods f o r Their Detection, Vol. 1, Plenum Press, New York-London, pp 267-282, 1971. Ames, B.N., Lee, F.D., and Durston, W.E. 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Dietary habits and cancer epidemiology. Cancer 43:1955-1961, 1979. Zaridze, D.G. Diet and cancer of the large bowel. Nutr. Cancer 2:241-249, 1981. APPENDIX A f e s t y l e Questionnaire 105 STUDY ON DIET AND FECAL MUTAGENS  QUESTIONNAIRE I. General I n f o r m a t i o n : Name; •  Age: .  Sex: B i r t h p l a c e : • •  Length of res idence i n Canada: P rev ious country of r e s i d e n c e : Length of t i m e : I I . Random 24-hour R e c a l l : P lease note on the form p r o v i d e d a l l foods and beverages c o n -sumed i n the p rev ious 24 hours . I n c l u d e the t ime consumed, the amount and the method o f p r e p a r a t i o n . Space i s p rov ided f o r r e c i p e s or e x p l a n a t i o n s . P lease record amounts i n e s t i m a t e d household measures; e . g . c u p , t a b l e s p o o n , teaspoon , ounces , number, e t c . P lease a l s o record m e d i c a t i o n s used d u r i n g t h i s 24 -hour p e r i o d : V i tamin/minera l supplement : •  Oral c o n t r a c e p t i v e : L a x a t i v e : •  P r e s c r i p t i o n drug (name) Other : I I I . T y p i c a l Day's Menu: P lease o u t l i n e a t y p i c a l d a y ' s food and beverage i n t a k e which you would be prepared t o f o l l o w d a i l y f o r 7 days . (The s p e c i f i c i tems may be s u b s t i t u t e d , but p r e p a r a t i o n methods must be the same, and foods must come from the same g roup . ) I n c l u d e snacks and o t h e r e x t r a s ( e . g . a l c o h o l ) on your o u t l i n e . 106 IV. S p e c i f i c I n f o r m a t i o n : 1. Do you t r i m the v i s i b l e f a t from meats? Always comple te l y trimmed -Always p a r t i a l l y trimmed _ _ _ _ _ Sometimes comple te l y trimmed Sometimes p a r t i a l l y trimmed ' Never 2. Do you use hot s p i c e s such as c h i l i e s , c u r r y , tabasco? Every day More than once per week More than once per month • R a r e l y or never • •• . 3.. Are you now f o l l o w i n g a s p e c i a l o r p r e s c r i b e d d i e t ? No Yes I f y e s , p l e a s e d e s c r i b e 4. Are you aware o f any major change i n your food s e l e c t i o n s ? In the past y e a r In the past 5 years I f y e s , p l e a s e d e s c r i b e 5 . Do you take any p i l l s o r m e d i c a t i o n s ? R e g u l a r l y O c c a s i o n a l l y At P r e s e n t Ora l c o n t r a c e p t i v e s N u t r i e n t supplements P r e s c r i p t i o n drugs (p lease name) 107 R e g u l a r l y O c c a s i o n a l l y At Present A s p i r i n Other (p lease name.) 6. Have you or any blood r e l a t i v e , ever had a medical d i a g n o s i s of any problem of the d i g e s t i v e t r a c t ? I f y e s , p l e a s e d e s c r i b e . 7. Have you o r any blood r e l a t i v e ever had a d i a g n o s i s of cancer? P l e a s e i n d i c a t e t ype . 8. How o f t e n do you u s u a l l y have a bowel movement? More than tw ice per day • Twice per day Every day Every 2 days • Every 3 days • Other ( s p e c i f y ) _ _ _ _ _ _ 9. Do you ever s u f f e r from c o n s t i p a t i o n ? / d i a r r h e a ? 10. Do you use l a x a t i v e s ? Every day -At l e a s t once per week O c c a s i o n a l l y Never 11 . Do you smoke? No. per day C i g a r e t t e s P ipe :. C iga rs Other • ' 108 12. Do you eve r consume a l c o h o l i c beverages? Yes : No: Wine Beer S p i r i t s No. o f d r i n k s per day No. o f d r i n k s per week No. o f ounces per d r i n k 109 DAILY FOOD RECORD (Use f o r 24-hour R e c a l l ) TIME NAME OF ITEM AMOUNT HOW PREPARED • Number of Bowel Movements: Use of M e d i c a t i o n s : COMMENTS, RECIPES AND EXPLANATIONS (use r e v e r s e i f necessary ) 110 DAILY FOOD RECORD (Use f o r T y p i c a l Day's Menu) TIME NAME OF ITEM AMOUNT HOW PREPARED Number of Bowel Movements: Use of M e d i c a t i o n s : COMMENTS, RECIPES AND EXPLANATIONS (use reve rse i f necessary ) APPENDIX B Food Frequency Questionnaire C N r H tH 2 3 FOOD ITEM More than once per day (note no. of times! MILK Homogenized Chocolate (comm.) 2% Skim (fresh or powdered) Evaporated Buttermilk YOGURT h c Plain - Homogenized 2% Skim F r u i t - Homogenized 2% Skim FROZEN DESSERTS Ice Cream Ice Milk Sherbet CREAM Sweet,sour or whipped Half and Half Non-dairy creamer Non-dairy whipped topping CHEESE Cottage cheese Cream cheese Other cheese H C h c 1 T Every day h c 1 ozl 1 oz! 5-6 times per week 2-4 times per week Once per week 3 times per i - month Twice per month Once per month Less than once per month FOOD ITEM More than once per day (note of time; Every day - mushrooms SjC - cauliflower I5C - broccoli *iC - greens '-iC - potato with skir 1 - potato without skin 1 - turnip I5C - onions *5C - beets jC - parsnips iC - brussel sprouts 6 - asparagus 6 - corn L or - eggplant IjC - peas *sC - beans-green or yellow !jC - beans-lima or broad >5C - leeks *sC - summer squash S5C - winter squash %C - other Dried - beans - peas ViC - l e n t i l s hC Vegetable j u i c e *5C 5-6 times per week 2-4 times per week Once per week 3 times P e r month Twice per month Once per month Less than once per month | Never • -FOOD ITEM More than once per day (note no. of timef Every day 5-6 times per week 2-4 times per week Once per week 3 times per month Twice per month Once per month Less than once per month Never Fruit j u i c e - c i t r u s - other VEGETABLES Raw - lettuce - cabbage - spinach - watercress - carrot - celery - tomato - cucumber - green onion - green or red pepper - mushrooms - cauliflower - broccoli - sprouts ( a l f a l f a bean, etc.) - other Cooked, Canned, Frozen - cabbage - spinach . - carrots - celery - tomato - pepper he hC >5C hC he hC he hC 1 k IT >SC He he he \c he he he he he he r LO iH FOOD ITEM More than once per day Cnote no. of timej Every day 5-6 tines per week 2-4 times per week Once per week • 3 times P e r month Twice per month Once per month Less than once per month • | Never - grapes - pineapple - strawberries - raspberries - blueberries - cherries - other Canned, Cooked, Frozen - orange - grapefruit - apple - pears - peach - apricot - grapes - pineapple - strawberries - raspberries - cherries- -. - f r u i t c o c k t a i l - other Dried - r a i s i n s - dates - f i g s - dried apples - dried apricots he he He he he he he he he he he he FOOD ITEM More than once per day (note no. of time Every day 5-6 times per week 2-4 times per week Once per week 3 times P e r month Twice per month Once per month Less than once per month Never FISH White f i s h Salmon S h e l l f i s h Canned f i s h - with o i l - without o i l Smoked f i s h [CEREALS P °4 Whole grain cerealsj Bran cereals Other cereals Whole grain breads Other breads Bran Bran baked products Brown r i c e Whole wheat pasta FRUITS Fresh - orange - grapefruit - apple - pear - banana - peach - apricot . - melon - avocado >5 c h c ! j C . s i . S i 1 h c t/4 d l l l l l l l 1 c h FOOD ITEM More than once per day (n°' e no. of timet Every day 5-6 times per week 2-4 times per week Once per week 3 times per month Twice per month Once per month Leas than once per month | Never VEAL Roas t Chops, steaks Cutlets, stew POULTRY 3 oz 3 oz 3 oz 2 oz 3 2 2 oz Chicken, Turkey Duck Goose GAME Deer Moose Other ORGAN MEATS Beef l i v e r Pork l i v e r Chicken l i v e r Heart Beef kidney Pork kidney Lamb kidney Tongue Other OTHER MEATS Bacon ( s l i c e s ) Sausages Luncheon meats (bologna,spiced meats, welners, etc.) FOOD ITEM More Chan once per day ( n ° t e  no. of time Every day 5-6 times per week 2-4 times per week EGGS Prepared with fat Prepared without fat In cooking FATS.OILS: (cooking or spreads^ Butter Margarine Vegetable o i l Lard Salad dressing Gravy BEEF Roast Steak Stew Hamburger (comm.) Ground beef Corned beef or Pastrami PORK Roast Chops, cutlets Ham LAMB Roast Chops Stew l-t-l 1 t 1 T 1 T 1 T 1 T 3 eel 3 oz 3 oz Once per week 3 times P e r month Twice per month Once per month Less than once per month Never -119 APPENDIX C Daily Food Record Form 120 DAILY FOOD RECORD TIME NAME OF ITEM AMOUNT HOW PREPARED -Number of Bowel Movements: Use of M e d i c a t i o n s : COMMENTS, RECIPES AND EXPLANATIONS (use r e v e r s e i f necessary ) 121 APPENDIX D Consent Form for P a r t i c i p a t i o n i n the Study CONSENT FORM STUDY ON DIET AND FECAL MUTAGENS I t has been suggested that c e r t a i n types of food may contribute to the r i s k of developing cancer of the colon. Diet most l i k e l y influences the s p e c i f i c types of b a c t e r i a found i n the gut, and some.bacteria may have the capacity to transform metabolites from some foods into p o t e n t i a l carcinogens. . The purpose of t h i s study i s to r e l a t e d i f f e r e n t d i e t a r y patterns to mutagenic a c t i v i t y i n the feces. We w i l l use the information and f e c a l samples which you provide f o r t h i s purpose. I f you agree to p a r t i c i p a t e i n the study, you w i l l be asked to: 1) Complete a questionnaire about some aspects of your d i e t , l i f e s t y l e , bowel habits and family h i s t o r y ; 2) Ingest a harmless dye so that your bowel t r a n s i t time can be established; 3) Follow a prescribed eating pattern f o r a period of f ive weeks; 4) Provide d a i l y records of your d i e t , medications and bowel ha b i t s . I, have had the purpose and requirements of t h i s study explained to me and I agree to p a r t i c i p a t e . I understand that any information which I provide w i l l be kept s t r i c t l y c o n f i d e n t i a l and that my name w i l l not appear i n any report concerning t h i s study. I am aware that I may withdraw from t h i s study at any time. (Signature) (Date) APPENDIX E Individual Dietary Guidelines. 124 Prescribed Daily Intakes # of Servings per Day Vegetarian Fruits/Vegetables 100% whole grain cereals Juice Vegetable oil/Margarine Eggs Milk products Meat as desired maximum 4T low fat none High Meat maximum 3 cups strained maximum 4T as desired high fat 3 oz. (cooked) lean beef + additional 3 oz. (cooked) lean red meat Ranges for consumption which are indicated on your prescription will ensure that all subjects have similar diets. Fiber Animal fat Vegetable fat Animal protein Vegetable protein Range of Daily Consumption Vegetarian High Meat {lh - 3x habitual) {h habitual maximum) {h habitual maximum) (2x habitual minimum) (lx habitual-4T maximum) (lx habitual-4T maximum) (h habitual maximum) (2x habitual minimum) (2x habitual minimum) (lx habitual maximum) NOTE: Basis for prescribed intakes appears in parentheses. APPENDIX F General Dietary Guidelines Guidelines for Diet Study The successful outcome of thi s study i s dependent on the vegetarian and high meat diets being as di f f e r e n t as possible. When selecting menus, please try to be "reasonably extreme" i n your choices so that we can better assess the effects of the two diets. It i s thought that the resul t s of our laboratory tests w i l l r e l a t e most closely to the amounts of f i b e r , red meat, and fat (both animal and vegetable) i n the diets. Therefore, based on a rough analysis of the habitual diet which you design, some Individual guidelines for the vegetarian and high meat diets w i l l be prescribed. During the vegetarian die t , please t r y to select foods which are high i n fi b e r and low i n animal f a t , and l i m i t your consumption of vegetable o i l s and margarine. The frequent use of legumes, nuts and seeds to replace animal proteins i s encouraged. In addition, use of low fat dairy products (milk, cheese, yogurt) w i l l help to reduce animal fat consumption. You w i l l be prescribed a minimi"*) number of servings of f r u i t s , vegetables and 100Z whole grain cereal products to consume daily during this period, as well as guidelines for consumption of f i b e r , f a t , and animal and vegetable proteins. During the high meat diet , please select foods which are low i n fib e r and high i n animal f a t . You w i l l be prescribed a maxim"") number of servings of f r u i t s , vegetables and j u i c e s , as well as guidelines for consumption of f i b e r , f a t , and animal and vegetable proteins. A minimum consumption of beef and other red meats w i l l be prescribed. Only refined cereal products should be consumed. Each day during the f i v e week study, please keep detailed records of your food consumption, bowel habits, and use of medications. Forms are provided for thi s purpose. General Guidelines Week 1 (7 days) Daily consumption of " t y p i c a l " menu which you have provided. Specific foods may be substituted i f they are of the same type and are prepared in the same way. Substitution l i s t s w i l l be provided. Weeks 2 and 3 (14 days) Vegetarian diet Please r e f r a i n completely from consuming any type of meat, f i s h or poultry. Low fat dairy products are permitted as outlined on your prescription. You are encouraged to u t i l i z e legumes, nuts and seeds i n place of animal proteins. Weeks 4 and 5 (14 days) High meat d i e t Please consume at least : 3 oz (cooked) of lean beef per day An additional 3 oz (cooked) of red meat per day (beef, lamb, pork, organ meats). The portions should consist of 3 oz lean meat, but you are not expected to purchase or consume lean meats only. Only refined cereals should be consumed. Individual prescriptions for consumption of foods during the vegetarian and high meat diets w i l l be provided. 127 APPENDIX G Diet Study Schedule Form 128 Diet Study Schedule Subject Week 1 - Habitual d i e t to (7 days) Fecal c o l l e c t i o n s . 2 days Use containers l a b e l l e d Use containers l a b e l l e d Weeks 2 and 3 - Vegetarian d i e t to ] (14 days) Fecal c o l l e c t i o n s c o l l e c t on 3 of these days Weeks 4 and 5 - High meat d i e t to (14 days) Fecal c o l l e c t i o n s ' c o l l e c t on 3 of these days Use containers l a b e l l e d 129 APPENDIX H Instructions for C o l l e c t i o n of Fecal Samples APPENDIX I Background Studies Using Urine, Serum and Feces to I n h i b i t Mutagenicity of Known Carcinogens Preliminary experiments were performed to assess the capacity of urine, serum and feces to trap and in a c t i v a t e the known carcinogens methyl nitro-nitroso-guadinine (MNNG) and 4-nitroquinoline-N-oxide (4NQ0). The re s u l t s of inte r a c t i o n s between mutagens and the i n t e r n a l environment, such as take place i n v i t r o i n these experiments, are us e f u l i n in t e r p r e t i n g the findings of other studies. In p a r t i c u l a r , the i n h i b i t o r y e f f e c t of aqueous extract of human feces on mutagenicity may be a factor i n the present d i e t study. The Ames test f o r mutagenicity was used i n these experiments. How-ever, i t was decided to use the f l u c t u a t i o n test i n the die t study since i t allows measurement of mutagenic a c t i v i t y with very low, non to x i c , concen-t r a t i o n s of f e c a l extract. On the evening preceding each experiment, overnight cultures were prepared by inoculating two tubes containing 5 ml nutrient broth with one drop of S&lmomZJta typhimusilum s t r a i n s TA 1535 and TA 100, and incubating overnight at 37 C to a concentration of 10 bac t e r i a I per ml. On the morning of the experiment, b a c t e r i a l subcultures were prepared by i n o c u l -ating 5 ml nutrient broth with 100 u 1 overnight culture, and incubating 4 hours at 37° C to a concentration of lG 7 8 /ml. The f e c a l extract was prepared by homogenizing feces with an equal weight of d i s t i l l e d water. A so l u t i o n c o n s i s t i n g of 0.2 ml homogenate and 1.8 ml phosphate buffered s a l i n e was f i l t e r e d , and the f i l t r a t e was retained. Urine and serum were used at 100% concentration. One ml aliquots of bac t e r i a subculture were centrifuged 5 minutes at 5,000 rpm. The supernatant was removed by suction, and the ba c t e r i a were suspended i n 1 ml of treatment medium (100% urine, 100% serum or 5% aqueous extract of feces with various concentrations of the carcinogens MNNG or 4NQ0, or d i s t i l l e d water). The bac t e r i a were incubated 20 minutes at 37* C, then centrifuged for 5 minutes at 5,000 rpm, washed by resuspension i n 1 ml 133 phosphate buffered s a l i n e , recentrifuged and resuspended i n 1 ml phosphate buffered s a l i n e . One hundred y l of treated b a c t e r i a i n suspension at a concentration of 10 8/ml was plated onto minimal agar plates, and 100 y l b a c t e r i a l sus-pension d i l u t e d to 10 3 /ml was plated onto nutrient agar plates. Three r e p l i c a t e s were plated for each sample. The plates were inverted and incub-ated 48 hours at 37° C. The number of colonies observed a f t e r 48 hours were averaged for each set of r e p l i c a t e s . The growth on minimal agar plates was used to ca l c u l a t e the number of revertant b a c t e r i a , and the growth on nutrient agar plates was used to c a l c u l a t e the numbers of bac t e r i a surviving the treatment. The number of revertant b a c t e r i a per 10 7 survivors was calculated and used to assess the capacity of each medium to trap and in a c t i v a t e the carcinogen. The r e s u l t s of these experiments appear i n Table 1.1. Urine and serum appear to trap the carcinogen MNNG at concentrations of 10 4M and 10 3M, and the carcinogen 4NQ0 at a concentration of 10 **M. Fecal extract appears to trap MNNG and 4NQ0 at concentrations of 5 10 3M. At the other concentrations of carcinogen, evaluation was not possible since the concen-t r a t i o n of carcinogen was either too low to e l i c i t a mutagenic response or too high, r e s u l t i n g i n i n h i b i t i o n of b a c t e r i a l growth or k i l l i n g i n a l l of the experiments. These r e s u l t s show that urine, serum and feces i n h i b i t mutagenicity of MNNG and 4NQ0. The mechanism of t h i s i n h i b i t i o n may be the r e s u l t of i n a c t i v a t i o n of the mutagen or of interference with the metabolic pathways which lead to mutations. Mutagens of i n t e r n a l o r i g i n might be i n h i b i t e d i n the body by s i m i l a r mechanisms. 134 TABLE 1.1 Number of Revertant Bacteria per 10 Survivors Carcinogen and Urine1 Serum Feces" concentration + Urine - Urine + Serum - Serum + Feces - Feces MNNG 5«10" 2M 192.1 445.0 10"3M 308.0 518.5 368.5 745.0 5*10~3M 1.4 328.9 10_ltM 1.0 12.7 1.9 364.2 S-IO^M 1.1 1.4 10~5M 1.3 0.9 1.3 1.7 0 1.4 1.4 1.7 1.4 1.0 2.3 4NQ0 5'10~2M K i l l i n g K i l l i n g 10~3M 400.0 I n h i b i t i o n 4 K i l l i n g K i l l i n g 5'10~3M 7.5 700.0 lO^M 31.0 697.7 302.3 1133.5 5'IO^M 8.9 8.7 10"5M 1.8 4.9 25.0 27.4 0 2.0 2.0 9.7. 8.3 7.1 5.7 '100% urine 2100% serum 35% aqueous f e c a l extract "*Formation of very small revertant colonies APPENDIX J Results of Experiments to E s t a b l i s h S t a b i l i t y of Fecal Mutag and Reproducibility of Fluctuation Test Results TABLE J . l Number of Fluctuation Test Tubes Containing Revertant Bacteria i n Repeated Fluctuation Tests on One Sample of Fecal Extract, Concentration 2.08yl/ml Date of Experiment TA 98 TA 100 24/10/79 37 31/10/79 36 07/11/79 32 19/12/79 35 05/11/79 24 12/12/79 26 18/12/79 21 Number of revertant tubes i n a t o t a l of 50 tubes. 137 APPENDIX K Individual Food Intakes of Subjects 1 to 6 on Baseline, Vegetarian and High Meat Diets TABLE K . l Daily Food Intakes by Number of Servings on Baseline Diet Subject Number 1 Nutrient Day Mean Range 1 2 3 h 5 6 71 Beef, 1 oz. 0.0 0.0 0.0 0.0 0.0 0.0 0.0 -Other meat, f i s h , poultry, 1 oz. U.O h.O h.O h.o h.o h.o h.O -Legumes, 1/2 cup 0.0 0.0 0.0 0.0 0.0 0.0 0.0 -Nuts and seeds, 1 tbsp. 2.0 2.0 2.0 2.0 2.0 2.0 2.0 -Whole milk products, 1 cup or equivalent 2.0 1.3 0.0 0.7 0.7 0.6 0.9 0.0-2.0 Reduced fat milk products, 1 cup or equivalent 0.5 0.5 1.1 0.5 0.5 1.1 0.7 0.5-1.1 Egg, 1 0.0 0.0 0.0 0.0 0.0 0.0 0.0 -Animal f a t , 1 tsp. 1.0 0.0 1.0 0.0 0.0 0.0 0.3 0.0-1.0 Vegetable o i l or margarine, 1 tsp. 0.0 1.0 1.0 1.0 0.0 0.0 0.5 0.0-1.0 Fruit/vegetable, 1/2 cup 6.2 6.5 5.0 h.5 5.0 h.5 5.3 5-6.5 Juice, 1/2 cup 1.5 1.5 1.5 1.5 1.5 1.5 1.5 -Whole grain c e r e a l , 1 exchange h.O h.5 h.5 3.5 h.5 3.5 h.l 3.5-U.5 Refined c e r e a l , 1 exchange 0.0 0.0 0.0 0.0 0.0 0.0 0.0 — Fecal sampling completed, and next d i e t commenced. TABLE K.2 Daily Food Intakes-by Number of Servings on Baseline Diet Subject Number 2 Nutrient Day • Mean Range 1 2 3 k 5 6 7 Beef, 1 oz. 0. 0 h.O 0.0 3. 0 0.0 0.0 1. 5 0.7 0. 0--h.o Other meat, f i s h , p o ultry, 1 oz. 3. 0 0.0 3.0 0. 0 3.0 h.O 0. 0 1.0 0. 0--h.o Legumes, 1/2 cup 0. 0 0.0 0.0 0. 0 0.0 0.0 0. 0 0.0 Nuts and seeds, 1 tbsp. 0. 0 0.0 0.0 0. 0 0.0 0.0 0. 0 0.0 Whole milk products, 1 cup 2. 0 2.0 2.0 3. 0 2.5 2.5 3. 0 2.8 2. 0--3.0 or equivalent Reduced fat milk products, 0. 3 0.1 1.0 0. 5 0.5 0.3 0. 5 0.5 0. 1--1.0 1 cup or equivalent Egg, 1 0. 0 0.0 0.0 0. 0 0.0 0.0 0. 0 0.0 Animal f a t , 1 tsp. 3. 0 9.0 3.0 2. 0 1.0 1.0 0. 0 3.2 0. 0--9.0 Vegetable o i l or margarine, 0. 0 0.0 0.0 0. 0 0.0 0.0 0. 0 0.0 1 tsp. Fruit/vegetable, 1/2 cup 6. 0 3.0 1+.8 5. 0 U.5 3.5 1. 5 h.l 1. 5--6.0 Juice, Il;/2ccup 0. 0 0.0 . 0.0 0. 0 0.0 0.0 0. 0 0.0 Whole grain c e r e a l , 1 exchange 2. 0 h.o 3.0 2. 0 2.0 3.0 2. 0 3.0 2. 0--h.o Refined c e r e a l , 1 exchange 0. 0 0.0 0.5 1. .0 0.5 0.3 3. 0 0.9 0. 0--3.0 TABLE K.3 Daily Food Intakes by Number of Servings on Baseline Diet Subject Number 3 Nutrient Day Mean Range 1 2 3 5 6 7 1 Beef, 1 oz. 0.0 0.0 0.0 3.0 5.0 0.0 1.3 0.0-5.0 Other meat, f i s h , poultry, 1 oz. 3.0 3.0 2.0 0.0 0.0 0.0 1.3 0.0-3.0 Legumes, 1/2 cup 0.0 0.0 0.0 0.0 0.0 0.0 0.0 -Nuts and seeds, 1 tbsp. 0.0 0.0 0.0 0.0 0.0 0.0 0.0 -Whole milk products, 1 cup or equivalent 2.8 2.5 3.2 3.5 1.5 2.8 2.7 1.5-3.5 Reduced f a t milk products, 1 cup or equivalent 0.5 0.2 0.5 0.3 0.3 0.3 O.h 0.2-0.5 Egg, 1 0.1 0.1 0.2 0.1 1.0 2.0 0.6 0.1-2.0 Animal f a t , 1 tsp. 1.0 2.0 0.0 h.O 0.0 0.0 1.2 0.0-U.O Vegetable o i l or margarine, 1 tsp. 3.0 2.0 6.0 2.0 5.0 3.0 3.5 2.0-6.0 Fruit/vegetable, 1/2 cup 5.5 5.0 2.5 h.5 3.5 3.8 U.l 2.5-5.5 Juice, 1/2 cup 0.5 0.5 1.0 0.5 1.0 0.5 0.7 0.5-1.0 Whole grain c e r e a l , 1 exchange 1.8 1.0 1.0 1.0 1.0 1.0 1.1 1.0-1.8 Refined c e r e a l , 1 exchange 0.5 1.0 1.0 1.0 1.0 3.0 1.3 0.5-3.0 Fecal sampling completed, and next diet commenced. TABLE K.h Daily Food Intakes by Number of Servings on Baseline Diet Subject Number h Nutrient Day Mean "Range . . . 1 . . 2 3 h 5 6 7 Beef, 1 oz. 3.5 0.0 3.5 0.0 0.0 h.O 0.0 1.6 0.0-H.O Other meat, f i s h , poultry, 1 oz. 0.0 h.O 0.0 3.5 h.o 0.0 3.0 2.1 0.0-k.O Legumes, 1/2 cup 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 -Nuts and seeds, 1 tbsp. 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 -Whole milk products, 1 cup or equivalent 1.0 1.0 1.0 1.0 1.0 1.0 1.5 1.1 1.0-1.5 Reduced f a t milk products, 1 cup or equivalent 0.5 0.5 0.5 0.5 0.5 0.5 0.7 0.5 0.5-0.7 Egg, 1 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 -Animal f a t , 1 tsp. 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.Q -Vegetable o i l or margarine, 1 tsp. 5.5 5.0 5.0 5.0 1.0 5.0 h.o h.h 1.0-5.5 Fruit/vegetable, 1/2 cup 8.5 6.0 6.0 7.0 6.5 6.0 5.0 6.h 5.0-8.0 Juice, 1/2 cup 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 -Whole grain c e r e a l , 1 exchange 2.2 2.3 2.3 2.2 2.0 • 3.5 3.0 2.5 2.0-3.5 Refined cereal., 1 exchange Q.O 0.0 Q.O 0.0 0.0 0.0 2.0 0.3 0.0-2.0 TABLE K.5 Daily Food Intakes by Number of Servings on Baseline Diet Subject Number 5 Day Nutrient Mean Range 1 2 3 h 5 6 T1 Beef, 1 oz. 0. 0 6. 0 0. 0 0. 0 6. 0 0 .0 2. 0 0. ,0--6.0 Other meat, f i s h , p o ultry, 1 oz. 5. 0 3. 0 9. 0 8. 0 3. 0 7. .0 5. 8 3. ,0--9.0 Legumes, 1/2 cup 0. 0 0. 0 0. 0 0. 0 0. 0 0 .0 0. 0 Nuts and seeds, 1 tbsp. 0. 0 0. 0 0. 0 0. 0 0. 0 0 .0 0. 0 Whole milk products, 1 cup 1. 0 0. 0 0. 0 0. 0 0. 0 0 .0 0. 2 0. ,0--1.0 or equivalent Reduced f a t milk products, 0. 0 0. 0 0. 3 0. 0 0. 2 0 .8 0. 2 0. ,0--0.8 1 cup or equivalent Egg, 1 2. 0 2.; 0 2. 0 2. 0 2. 0 2 .0 2. 0 Animal f a t , 1 tsp. 3. 0 0. 0 0. 0 0. 0 6. 0 0 .0 1. 5 0. .0--6.0 Vegetable o i l or margarine, 5. 0 9. 0 6. 0 10. 0 9. 0 8 .0 7. 8 5. .0--10.0 1 tsp. Fruit/vegetable, 1/2 cup. h. 0 3. 0 3. 5 h. 0 h. 5 h .0 3. 8 3. .0-Juice, 1/2 cup 1. 0 1. 0 1. 0 1. 0 1. 0 1 .0 1. 0 Whole grain c e r e a l , 1 exchange 6. 0 7. 0 6. 0 7. 0 5. 0 7 .0 6. 3 5 .0--7.0 Refined c e r e a l , 1 exchange 0. 0 0. 0 0. 3 0. 0 It. 0 0 .2 0. 8 0, .0--h.o Fecal sampling completed, and next diet commenced. TABLE K.6 Daily Food Intakes by Number of Servings on Baseline Diet Subject Number 6 Day Nutrient Mean .Range 1 2 3 h 5 6 T 1 Beef, 1 oz. 0.0 h.O 0.0 0.0 7.0 0.0 1.8 0.0-7.0 Other meat, f i s h , p o ultry, 1 oz. 3.0 3.0 5.0 8.0 2.0 8.0 U.8 2.0-8.0 Legumes, 1/2 cup 0.0 0.0 0.0 1.0 0.0 0.0 0.2 0.0-1.0 Nuts and seeds, 1 tbsp. 0.0 0.0 0.0 0.0 0.0 0.0 0.0 -Whole milk products, 1 cup or equivalent 0.0 0.0 0.0 0.0 0.0 0.0 0.0 -Reduced fat milk products, 1 cup or equivalent 1.3 1.3 1.0 1.5 1.0 1.5 1.3 1.0-1.5 Egg, 1 1.0 1.0 2.0 2.0 3.0 2.0 1.8 1.0-3.0 Animal f a t , 1 tsp. 0.0 0.0 0.0 0.0 1.0 0.0 0.2 0.0-1.0 Vegetable o i l or margarine, 1 tsp. 6.0 8.0 h.o 6.0 10.0 10.0 7.3 U.0-10. Fruit/vegetable, 1/2 cup 5.0 7.5 5.0 7.0 5.5 6.0 6.0 5.0-7.5 Juice, 1/2 cup 1.0 1.0 1.0 1.0 1.0 1.0 1.0 -Whole grain c e r e a l , 1 exchange 3.0 1.0 3.0 3.0 1.0 2.. 5 2.3 1.0-3.0 Refined c e r e a l , 1 exchange 0.0 0.0 0.0 0.0 1.5 0.0 0.3 0.0-1.75 . Fecal sampling completed, and next diet commenced. TABLE K.7 Daily Food Intakes by Number of Servings on Vegetarian Diet Subject Number 1 Day 1 2 3 1* 5 6 7 8 9 10 11 12 13 Mean l i t 1 Range Beef, 1 oz. 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 Other meat, f i s h , poultry, 1 oz. 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 Legumes, 1/2 cup 1.0 2.0 1.0 2.5 0.0 1.0 2.0 0.0 0.0 2.0 2.0 1.5 0.0 1.2 0.0-2.5 Nuts and seeds, 1 tbsp. 2.0 6.0 2.0 6.0 2.0 5.0 1.0 10.0 0.3 0.0 U.O 0.0 6.0 2.6 0.0-10.0 Whole milk products, 1 cup or equivalent 0.0 1.2 0.0 .0.5 0.0 0.0 0.0 0.0 0.0 0.0 2.0 0.6 0.0 0.3 0.0-1.2 Reduced f a t milk products, 1 cup or equivalent 1.2 1.5 2.3 1.5 1.8 0.5 1.1* 1.1 1.0 1.5 2.5 0.0 0.5 1.3 0.0-2.5 Egg, 1 0.0 0.1 0.0 0.3 0.0 0.1 0.0 0.0 0.0 0.0 0.1 0.0 1.3 0.1 0.0-1.3 Animal f a t , 1 tsp. 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 _ Vegetable o i l or margarine, 1 tsp. 1.0 5.0 1.0 3.5 1.0 2.0 2.0 0.0 0.0 1.5 3.0 1.0 5.0 2.0 0.0-5.0 Fruit/vegetable, 1/2 cup 8.5 11.0 8.5 6.5 h.O 11.5 5.5 9.0 11.5 5.5 lt.0 7.5 • 1.0 7.2 1.0-11.5 Juice, 1/2 cup 0.0 1.5 0.0 0.0 2.0 0.0 1.5 2.0 0.0 1.0 1.0 0.0 0.0 0.7 0.0-2.0 Whole grain c e r e a l , 1 exchange 6.0 6.5 h.O lt.0 3.5 7.5 6.0 5.5 6.0 i*.o 5.0 5.0 i*.o 5.2 It.0-7.5 Refined c e r e a l , 1 exchange 0.0 0.5 0.0 0.0 0.0 0.0 1.0 1.0 0.0 0.3 0.3 0.3 0.0 0.3 0.0-1.0 1 Fecal sampling completed, and next d i e t commenced. TABLE K.8 Daily Food Intakes by Number of Servings on Vegetarian Diet Subject Number 2 Day Nutrient 1 2 3 It 5 6 7 8 9 10 11 12 13 ••' Mean Range Beef, 1 oz. 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 -Other meat, f i s h , poultry, 1 oz. 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 -Legumes, 1/2 cup 0.0 1.0 0.0 0.0 0.0 0.0 0.0 0.0 1.0 0.0 1.0 0.0 0.0 0.2 0.0-1.0 Nuts and seeds, 1 tbsp. 0.0 0.0 0.0 0.0 0.0 . 0.5 0.0 0.0 3.0 0.0 8.0 . 0.0 0.0 0.9 0.0-8.0 Whole milk products, 1 cup or equivalent 2.0 1.5 0.5 1.0 1.0 0.5 1.8 2.0 1.0 h.O ' 2.0 2.0. 3.0 1.7 0.5-it.O Reduced fat milk products, 1 cup or equivalent 0.0 1.0 0.5 0.5 0.8 0.3 1.0 1.5 0.8 0.2 0.5 0.5 0.3 0.6 0.0-1.0 Egg, 1 0.0 0.0 1.5 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.1 0.0-1.5 Animal f a t , 1 tsp. 0.0 0.0 0.0 0.0 0.0 9.0 0.0 0.0 3.0 6.0 0.0 0.0 0.0 1.1* 0.0-9.0 Vegetable o i l or margarine, 1 tsp. 3.0 1.0 0.0 6.0 3.0 h.o 3.0 0.0 0.0 0.0 6.0 0.0 1+.0 2.3 0.0-6.0 Fruit/vegetable, 1/2 cup 6.0 h.5 6.0 5.0 7.0 U.5 7.8 7.5 3.2 9.0 7.0 3.5 h.O 5.8 3.2-9.0 Juice, 1/2 cup 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 -Whole grain c e r e a l , 1 exchange 3.0 3.0 5.0 5.0 h.5 h.O 3.5 3.5 3.5 5.0 h.O 5.0 lt.0 h.l 3.0-5.0 Refined c e r e a l , 1 exchange 0.5 0.0 0.0 0.3 0.0 0.5 0.0 0.5 1.5 0.0 0.0 0.0 1.0 0.3 0.0-1.5 Fecal sampling completed, and next diet commenced. TABLE K.9 Daily Food Intakes by Number of Servings on Vegetarian Diet. Subject Number 3 Day Nutrient 1 2 3 It 5 6 7 8 9 10 11 12 13 1 Mean l i t 1 Range Beef, 1 oz. 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 -Other meat, f i s h , poultry, 1 oz. 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 -Legumes, 1/2 cup 0.0 0.0 0.0 1.5 0.0 2.0 0.5 0.5 0.0 2.0 0.0 0.0 0.5 0.0-2.0 Nuts and seeds, 1 tbsp. 0.0 2.0 0.0 0.0 lt.0 0.0 12.0 lt.0 0.0 8.0 lt.0 lt.0 3.2 0.0-12.0 Whole milk products, 1 cup or equivalent 2.0 1.5 1.0 1.5 2.5 1.0 1.0 3.0 2.5 2.3 1.5 3.5 1.9 1.0-3.5 Reduced fat milk products, 1 cup or equivalent 0.5 1.0 1.0 0.8 0.2 1.3 0.7 1.0 0.8 0.5 0.5 O.lt 0.7 0.2-1.0 Egg, 1 2.0 0.5 0.0 0.1 0.0 0.0 0.0 2.0 0.0 0.0 0.0 0.0 O.lt 0.0-2.0 Animal f a t , 1 tsp. 0.0 2.0 0.0 1.0 2.0 3.0 0.0 0.0 0.0 0.0 2.0 6.0 1.3 0.0-6.0 Vegetable o i l or margarine, 1 tsp. 7.0 1.0 lt.0 1.0 0.0 lt.0 1.0 2.0 7.0 0.0 1.0 0.0 2.3 0.0-7.0 Fruit/vegetable, 1/2 cup 5.5 5.5 5.0 it.5 6.3 2.5 3.5 3.5 3.8 lt.0 2.5 3.5 It.2 2.5-6.3 Juice, 1/2 cup 0.5 0.5 0.5 1.0 0.5 0.0 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.0-1.0 Whole grain c e r e a l , 1 exchange 2.5 lt.0 3.0 6.0 lt.0 8.0 5.0 3.0 6.0 3.7 5.5 lt.0 It.5 2.5-8.0 Refined c e r e a l , 1 exchange 0.0 1.5 0.5 1.0 0.0 1.0 1.0 0.0 1.0 1.5 1.0 3.0 1.0 0.0-3.0 Fecal sampling completed, and next d i e t commenced. TABLE K.10 Daily Food Intakes by Number of Servings on Vegetarian Diet Subject Number h Nutrient Day Range 1 2 3 1* 5 6 7 8 9 10 11 12 13 • 1 " Mean IM Beef, 1 oz. 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 -Other meat, f i s h , poultry, 1 oz. 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 -Legumes, 1/2 cup 0.3 0.0 0.0 0.0 0.0 0.0 0.5 0.0 0.0 0.0 0.0 0.0 2.0 0.2 0.0-2.0 Nuts and seeds, 1 tbsp. 0.0 3.0 5.0 2.0 2.0 6.0 1.0 3.0 2.0 2.0 0.0 0.0 0.0 2.0 0.0-6.0 Whole milk products, 1 cup or equivalent 0.5 1.0 2.0 0.5 1.5 1.5 0.5 1.2 0.0 2.0 2.0 2.0 0.0 1.1 0.0-2.0 Reduced f a t milk products, 1 cup or equivalent 0.8 0.5 0.5 1.5 1.0 0.5 0.3 0.8 1.7 2.0 1.0 1.5 1.3 1.0 0.3-2.0 Egg, 1 0.5 1.0 1.0 1.0 0.0 0.5 1.0 1.0 0.0 2.0 1.0 1.0 0.0 0.8 0.0-2.0 Animal f a t , 1 tsp. 0.0 0.0 0.0 1.0 1.0 0.0 0.0 0.0 0.0 0.0 5.0 1.0 0.0 0.6 0.0-5.0 Vegetable o i l or margarine, 1 tsp. 2.0 1.0 5.0 7.0 5.0 3.0 2.0 3.0 2.0 l t .0 5.0 1.0 1.0 3.2 1.0-7.0 Fruit/vegetable, 1/2 cup 6.0 6.5 9.5 8.0 8.5 8.0 6.5 7.5 l t .0 6.0 •6.5 5.0 5.3 6.7 lt.0-9-5 Juice, 1/2 cup 0.0 0.0 0.3 2.0 2.0 0.5 0.5 2.0 2.0 2.0 2.0 2.0 0.0 1.2 0.0-2.0 Whole grain c e r e a l , 1 exchange 4.0 6.0 l t .0 5,0 5.0 6.0 3.0 l t .0 5.5 3.0 5.0 It.3 0.0 It.2 0.0-6.0 Refined c e r e a l , 1 exchange 0.0 0.Q 0.0 0.0 0.0 0.0 1.0 0.0 0.0 0.5 0.0 0.0 0.0 0.1 0.0-1.0 1 Fecal sampling completed, and next d i e t commenced. TABLE K . l l Daily Food Intakes by Number of Servings on Vegetarian Diet Subject Number 5 Day Nutrient 1 2 3 4 5 6 7 8 9 10 11 12 13 Mean 141 Range Beef, 1 oz. 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 -Other meat, f i s h , poultry, 1 oz. 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 -Legumes, 1/2 cup 0.0 0.0 2.0 2.0 1.0 0.0 2.0 4.0 0.0 4.0 1.0 2.0 0.0 1.4 0.0-4.0 Nuts and seeds, 1 tbsp. 0.0 4.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 2.0 1.0 0.0 0.5 0.0-4.0 Whole milk products, 1 cup or equivalent 0.0 0.6 1.5 0.5 1.0 1.5 0.0 0.0 1.5 0.0 1.0 0.0 1.5 0.7 0.0-1.5 Reduced f a t milk products, 1 cup or equivalent ~0.0 0.5 2.5 0.0 1.0 1.0 2.0 1.0 0.6 1.0 1.3 0.5 0.0 0.9 0.0-2.0 Egg, 1 2.0 0.0 2.0 2.0 0.0 1.0 1.0 0.0 0.0 0.0 0.0 1.0 0.5 0.7 0.0-2.0 Animal f a t , 1 tsp. 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 3.0 0.2 0.0-3.0 Vegetable o i l or margarine, 1 tsp. 6.0 14.0 2.0 7.0 2.0 2.0 0.0 3.0 15.0 1.0 5.0 9.0 5.0 5.5 0.0-15.0 Fruit/vegetable, 1/2 cup 6.5 5.5 2.5 5.5 7.5 2.5 8.0 5.0 8.5 5.5 5.0 10.5 9.0 6.3 2.5-10.5 Juice, 1/2 cup 1.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.0 0.0 0.0 1.0 0.0 0.2 0.0-1.0 Whole grain c e r e a l , 1 exchange 6.5 6.5 5.0 5.0 2.5 6.5 4.0 5.0 6.0 5.0 5.5 6.5 3.0 5.2 3.0-6.5 Refined c e r e a l , 1 exchange 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 3.0 0.0 0.0 0.0 1.5 0.3 0.0-3.0 Fecal sampling completed, and next d i e t commenced. TABLE K.12 Daily Food -Intakes by Number of Servings on Vegetarian Diet Subject Number 6 Day Nutrient 1 2 3 1* 5 6 7 8 9 10 11 12 Mean 13 14 1 Range Beef, 1 oz. 0.0 0.0 0.0 0.0 0. 0 0.0 0.0 0. 0 0.0 0.0 0.0 0.0 0.0 0.0 Other meat, f i s h , poultry, 1 oz. 0.0 0.0 0.0 0.0 0. 0 0.0 0.0 0. 0 0.0 0.0 0.0 0.0 0.0 0.0 Legumes, 1/2 cup 0.0 0.0 0.5 1.5 0. 0 0.0 2.0 2. 0 0.0 2.0 0.0 0.0 0.0 0.6 0.0--2.0 Nuts and seeds, 1 tbsp. 0.0 2.0 0.0 0.0 0. 0 0.0 0.0 0. 5 0.0 0.0 0.0 0.0 0.0 0.2 0.0--2.0 Whole milk products, 1 cup 0.0 0.0 1.5 0.0 1. 5 0.0 0.5 0. 0 1.0 0.5 0.0 3.0 0.0 0.6 0.0--3.0 or equivalent Reduced f a t milk products, 1.0 3.0 2.0 3.0 2. 0 2.3 2.5 1. 5 2.5 1.0 3.0 3.0 2.0 2.2 1.0--3.0 1 cup sor equivalent Egg, 1 2.0 2.0 2.0 1.0 0. 0 1.0 1.0 0. 0 0.0 0.0 1.0 1.0 1.2 0.9 0.0--2.0 Animal f a t , 1 tsp. 0.0 0.0 3.0 0.0 0. 0 0.0 0.0 0. 0 0.0 0.0 0.0 0.0 4.0 0.5 0.0--4.0 Vegetable o i l or margarine, 7.0 15.0 1.0 5.0 2. 0 4.5 0.0 3. 0 11.0 2.0 6.0 8.0 4.0 5.3 0.0--15.0 1 tsp. Fruit/vegetable, 1/2 cup 6.5 6.5 6.5 7.5 5. 5 3.0 10.0 7. 0 8.0 10.5 7.0 11.0 7.5 7.4 3.0--11.0 Juice, 1/2 cup 1.0 1.5 O'.O 0.0 0. 0 0.0 0.0 1. 5 1.0 1.0 0.0 1.0 1.5 0.7 0.0--1.5 Whole grain c e r e a l , 1 exchange 4.5 7.5 3.5 4.0 1*. 5 7.3 4.0 4. 0 6.0 1.5 5.5 4.0 0.0 4.3 0.0--7.5 Refined c e r e a l , 1 exchange 0.0 0.0 0.0 0.0 0. 0 0.0 0.0 0. 0 3.0 0.0 0.0 0.0 1.5 0.3 0.0--3.0 Fecal_ sampling completed, and next diet commenced. TABLE K.13 Daily Food Intakes by Number of Servings on High Meat Diet Subject Number 1 Nutrient Day Range 1 2 3 1+ 5 6 7 8 9 10 11 12 13 Mean i i * 1 Beef, 1 oz. 6.0 3.0 6.0 6.0 3.0 6.0 3.0 3.0 3.0 3.0 3.0 8.0 3.0 It.3 3.0-8.0 Other meat, f i s h , poultry, 1 oz. 0.0 3.0 0.0 0.0 3.0 2.0 3.0 3.0 3.0 3.0 3.0 2.0 3.0 2.2 0.0-3.0 Legumes, 1/2 cup 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 -Nuts and seeds, 1 tbsp. 0.0 0.0 0.0 U.O U.O 0.0 0.0 0.0 0.0 0.0 lt.0 0.0 1.0 1.0 0.0-lt.O Whole milk products, 1 cup or equivalent 0.0 0.0 0.0 1.0 0.0 1.0 0.6 1.0 0.9 0.8 0.3 1.0 0.0 0.5 0.0-1.0 Reduced fat milk products, 1 cup or equivalent 1.0 1.0 0.8 1.5 0.0 0.8 0.0 0.5 0.5 0.8 0.0 0.0 0.5 0.7 0.0-1.5 Egg. 1 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.3 0.3 0.2 0.0 0.0 0.1 0.0-0.3 Animal f a t , 1 tsp. 0.0 0.0 0.0 1.0 0.0 2.0 0.0 0.0 7.0 7.0 3.0 0.0 0.0 1.5 0.0-7.0 Vegetable o i l or margarine, 1 tsp. 3.0 1.0 0.0 2.0 5.0 lt.0 2.0 lt.0 3.0 2.0 lt.0 5.0 0.0 2.7 0.0-5.0 Fruit/vegetable, 1/2 cup 1.5 1.5 5.5 3.0 2.5 it.5 U.5 3.5 1.0 It.5 lt.0 2.5 lt.0 3.3 1.0-5.5 Juice, 1/2 cup 8.0 5.5 1.0 1.0 1.0 It.5 2.0 0.0 0.0 2.0 0.0 3.5 2.0 2.3 0.0-8.0 Whole grain c e r e a l , 1 exchange 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 -Refined c e r e a l , 1 exchange 3.0 3.0 i*.o 6.5 6.0 5.0 5.0 3.0 9.0 5.0 3.0 5.5 5.0 It.8 3.0-9.0 !Fecal sampling completed, and diet discontinued. TABLE K . l4 Daily Food Intakes by Number of Servings on High Meat Diet Subject Number 2 Nutrient Day Range 1 2 3 It 5 6 7 8 9 10 11 12 13 lit Beef, 1 oz. 5.0 lt.0 6. 0 6.0 6.0 6.0 7.0 6.0 3.0 4.0 5.0 6.0 7.5 6.0 5.5 3.0--7.5 Other meat, f i s h , poultry, 1 oz. 2.0 2.0 2. 0 0.0 0.0 0.0 1.0 0.0 2.0 2.0 2.0 1.5 0.0 0.0 1.0 0.0--2.0 Legumes, 1/2 cup 0.0 0.0 0. 0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 Nuts and seeds, 1 tbsp. 0.0 0.0 0. 0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 Whole milk products, 1 cup 1.5 3.0 1. 5 2.5 3.5 2.5 1.5 2.0 2.3 3.0 2.2 2.0 "• • 1.0 0.5 2.1 0.5--3.5 or equivalent Reduced fat milk products, 0.3 0.3 0. 0 0.3 0.2 0.5 0.5 0.5 0.3 0.3 0.2 0.3 0.2 0.5 0.3 0.0--0.5 1 cup or equivalent Egg, 1 0.0 0.0 0. 0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 Animal fat, 1 tsp. 2.0 3.0 7. 0 3.0 3.0 0.0 0.0 4.0 0.0 6.0 8.0 6.0 6.0 3.0 3.6 0.0--8.0 Vegetable o i l or margarine, 6.0 6.0 3. 0 12.0 0.0 0.0 0.0 0.0 6.0 9.0 3.0 9.0 3.0 0.0 4.1 0.0--12.0 1 tsp. Fruit/vegetable, 1/2 cup 5.0 4.5 3. 0 1.0 4.5 4.0 2.0 4.0 2.0 3.5 4.0 1.0 2.8 1.0 3.0 1.0--5.0 Juice, 1/2 cup 1.0 1.0 0. 0 1.0 1.0 0.0 0.0 0.0 0.0 0.0 1.0 1.0 0.0 1.0 0.5 0.0--1.0 Whole grain c e r e a l , 1 exchange 0.0 0.0 0. 5 0.0 2.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.2 0.0--2.0 Refined c e r e a l , 1 exchange 1.5 2.0 It. 5 5.0 3.5 3.0 4.0 4.0 6.0 6.5 3.0 4.0 5.5 2.0 3.9 1.5--6.5 TABLE K.15 Daily Food Intakes "by Number of Servings on High Meat Diet Subject Number 3 Day Nutrient 1 2 3 It 5 6 7 8 9 10 11 12 13 lit Mean Range Beef, 1 oz. 6.0 3.0 5.0 6.0 8.0 6.5 5.0 7.0 7.0 7.0 7.0 6.0 6.0 6.0 6.1 3.0-8.0 Other meat, f i s h , poultry, 1 oz. 0.0 5.0 0.0 3.0 0.0 0.0 5.0 0.0 1.0 0.0 0.0 0.0 0.0 0.0 1.0 0.0-5.0 Legumes, 1/2 cup 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 -Nuts and seeds, 1 tbsp. 0.0 2.0 0.0 0.0 0.0 0.0 lt.0 0.0 0.0 lt.0 3.0 0.0 3.0 1.0 1.2 0.0-lt.O Whole milk products, 1 cup or equivalent 1.3 1.0 2.5 1.0 0.5 2.2 1.5 2.2 1.0 0.8 1.5 3.8-. 3.0 1.0 1.7 0.5-3.8 Reduced f a t milk products, 1 cup or equivalent 0.3 0.5 0.0 0.0 0.0 0.0 0.5 0.2 0.3 0.3 0.0 0.5 0.3 0.3 0.2 0.0-0.5 Egg, 1 0.0 0.0 0.0 0.0 0.0 0.0 0.5 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0-0.5 Animal f a t , 1 tsp. lt.0 0.0 6.0 0.0 0.0 0.0 6.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.1 0.0-6.0 Vegetable o i l or margarine, 1 tsp. 6.0 9.0 0.0 5.0 3.0 2.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 8.0 2.It 0.0-9.0 Fruit/vegetable, 1/2 cup 6.0 5.5 2.0 it.5 •3.0 2.0 2.0 1.5 2-5 3.0 . 3.2 lt.0 1.8 lt.0 3.2 1.5-6.0 Juice, 1/2 cup 3.5 2.0 0.0 0.0 0.5 1.5 1.0 3.5 0.5 2.0 0.0 0.5 0.0 0.0 1.1 0.0-3.5 Whole grain c e r e a l , 1 exchange 1.0 1.0 1.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.2 0.0-1.0 Refined c e r e a l , 1 exchange 2.5 3.0 5.0 5.0 7.0 lt.0 6.5 8.0 6.0 . 5.0 5.5 3.5 6.0 7.0 5.3 2.5-8.0 TABLE K.l6 Daily Food Intakes by Number of Servings on High Meat Diet Subject Number 4 Day Nutrient 1 2 3 1* 5 6 7 8 9 10 11 12 13 Mean 141 Range Beef, 1 oz. 6.0 6.0 6.0 6.0 .6.0 6.0 4.0 6.0 7.0 3.0 6.0 6.0 6.0 5.7 3.0-7.0 Other meat, f i s h , poultry, 1 oz. 0.0 2.0 1.0 0.0 0.0 0.0 3.0 0.0 0.0 3.0 0.0 0.0 0.0 0.7 0.0-3.0 Legumes, 1/2 cup 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 -Nuts and seeds, 1 tbsp. 1.0 0.0 1.0 1.0 0.0 0.0 1.0 0.0 0.0 0.0 1.0 1.0 0.0 0.5 0.0-1.0 Whole milk products, 1 cup or equivalent 0.5 0.5 0.5 0.5 0.5 0.0 0.5 2.0 0.5 0.5 0.0 0.0 1.0 0.5 0.0-2.0 Reduced fat milk products, 1 cup or equivalent 0.2 1.0 1.0 0.5 0.5 0.5 1.0 1.5 1.0 0.3 1.0 1.2 0.5 0.8 0.2-1.0 Egg, 1 1.0 0.0 0.0 1.0 0.0 0,0 0.0 1.0 1.0 0.0 1.0 1.0 2.0 0.6 0.0-2.0 Animal f a t , 1 tsp. 2.0 2.0 4.0 1.0 9.0 h.O 9.0 0.0 3.0 2.0 11.0 11.0 2.0 4.6 0.0-11.0 Vegetable o i l or margarine, 1 tsp. 5.0 2.0 1.0 h.O 2.0 0.0 0.0 3.0 2.0 0.0 2.0 6.0 3.0 2.3 0.0-6.0 Fruit/vegetable, 1/2 cup 3.0 2.5 3.5 • h.O 2.5 3.5 4.5 2.0 3.0 3.2 3.0 4.0 2.3 3.2 2.0-4.5 Juice, 1/2 cup 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 -Whole grain c e r e a l , 1 exchange 0.0 0.0 0.0 0.0 0.0 1.0 0.0 0.0 0.0 0.0 0.0 0.0 2.0 0.2 0.0-2.0 Refined c e r e a l , 1 exchange 3.5 5.0 4.0 4.0 5.0 5.0 4.5 3.0 3.0 2.0 5.0 5.5 3.0 4.0 2.0-5.5 Fecal sampling completed, and diet discontinued. TABLE K.17 Daily Food Intakes "by Number of Servings on High Meat Diet Subject Number 5 Nutrient Day Range 1 2 3 It 5 ( S 7 8 9 10 11 12 13 1 Mean IU1 Beef, 1 oz. 11.0 9.0 7.0 6.0 lt.0 3.0 3.0 6.0 lt.0 lt.0 5.0 lt.0 8.0 5.7 3.0-11.0 Other meat, f i s h , poultry, 1 oz. 0.0 0.0 0.0 2.0 • 3.0 6.0 lt.0 lt.0 It.5 5.0 5.0 lt.0 3.0 3.1 0.0-6.0 Legumes, 1/2 cup 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 1.0 0.0 0.0 0.0 0.0 0.1 0.0-1.0 -Nuts and seeds, 1 tbsp. 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 -Whole milk products, 1 cup or equivalent 1.0 0.0 0.2 1.0 0.0 3.0 0.0 0.0 0.0 1.2 0.3 0.0 1.0 0.6 0.0-3.0 Reduced fat milk products, 1 cup or equivalent 0.1 0.5 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 <0.1 0.0-0.5 Egg, 1 2.0 0.0 2.0 0.0 1.0 2.0 2.0 2.0 2.0 0.0 0.0 2.0 0.0 1.6 0.0-2.0 Animal f a t , 1 tsp. 9.0 0.0 0.0 6.0 0.0 0.0 3.0 0.0 3.0 0.0 0.0 0.0 1.0 1.7 0.0-9.0 Vegetable o i l or margarine, 1 tsp. 3.0 2.0 11.0 6.0 6.0 7.0 lt.0 3.0 7.0 lt.0 0.0 6.0 5.0 It.9 0.0-11.0 Fruit/vegetable, 1/2 cup 7.0 2.5 3.5 8.0 3.0 5.0 lt.0 6.0 5.0 6.0 8.0 5.5 It.5 5.2 2.5-8.0 Juice, 1/2 cup 1.0 1.0 0.0 1.0 0.0 0.0 1.5 0.0 0.0 0.0 0.0 0.0 0.0 O.lt 0.0-1.5 Whole grain c e r e a l , 1 exchange 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 -Refined c e r e a l , 1 exchange 7.0 7.5 6.0 2.0 6.0 5.5 8.3 5.0 7.0 lt.0 7.0 6.0 lt.0 5.8 2.0-8.3 1 Fecal sampling completed, and diet discontinued. TABLE K.18 Daily Food Intakes by Number of Servings on High Meat Diet Subject Number 6 Day Nutrient 1 2 3 4 5 6 7 8 9 10 11 12 Mean 13 14 1 Range Beef, 1 oz. 7.0 8. 0 6. 0 6.0 3.0 3. 0 3.0 6.0 4.0 3.0 3.0 3.0 7.0 4.8 3. 0--8.0 Other meat, f i s h , poultry, 1 oz. 0.0 0. 0 0. 0 3.0 3.0 it. 0 4.o 3.0 4.0 4.0 3.0 3.0 2.0 2.5 0. 0--4.0 Legumes, 1/2 cup 0.0 0. 0 0. 0 0.0 0.0 0. 0 0.0 0.0 1.0 0.0 0.0 0.0 0.0 0.1 0. 0--1.0 Nuts and seeds, 1 tbsp. 0.0 0. 0 0. 0 0.0 0.0 0. 0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 Whole milk products, 1 cup 1.1 0. 8 0. 2 3.0 0.0 2. 0 0.5 1.0 0.0 1.5 0.5 1.0 1.2 1.0 0. 0--3.0 or equivalent Reduced fat milk products, 0.0 0. 0 0. 0 0.0 0.0 0. 0 0.0 0.0 0.0 0.0 0.0 O.'O 0.0 0.0 1 cup or equivalent Egg, 1 2.0 0. 0 1. 0 0.0 1.0 1. 0 1.5 2.0 1.0 0.0 0.0 1.0 0.0 0.8 0. 0--2.0 Animal f a t , 1 tsp. 1.0 0. 0 0. 0 2.0 0.0 3. 0 2.0 0.0 2.0 0.0 0.0 4.0 2.0 1.2 0. 0--4.0 Vegetable o i l or margarine, 3.0 2. 0 6. 0 5.0 5.0 It. 0 U.o 2.0 5.0 2.0 0.0 6.0 6.0 3.1 0. 0--6.0 1 tsp. Fruit/vegetable, 1/2 cup 5.5 3. 5 3. 5 5.5 3.5 5. 0 3.0 6.0 5.0 5.0 6.0 3.0 4.5 4.5 3. 0--6.0 Juice, 1/2 cup 1.0 1. 0 0. 0 1.0 0.0 0. 0 1.5 0.0 0.0 0.0 0.0 0.0 0.0 0.3 0. 0--1.5 Whole grain c e r e a l , 1 exchange 0.0 0. 0 0. 0 0.0 0.0 0. 0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 Refined c e r e a l , 1 exchange 5.0 5. 0 6. 5 1.0 4.5 6. 0 4.5 2.5 4.0 5.0 5.0 4.0 3.0 4.3 1. 0--6.5 Fecal sampling completed, and diet discontinued. 156 APPENDIX L Fluctuation Test Results for Individual Fecal Samples TABLE L . l Number of Fluctuation Test Tubes Containing Revertant Bacteria A f t e r 5 Days, Subject Number 1 Salmonella s t r a i n TA 98 Sample Extract concentration (ul/ml) 8.33 4.17 2.08 1.04 0.52 0.0 B l 1 38 37 34 35 25 17 B2 50 41 36 29 30 19 VI 2 40 42 35 27 21 17 V2 50 38 40 32 27 18 V3 33 34 38 35 34 12 HI 3 50 38 42 32 27 14 H2 29 26 26 28 24 11 H3 49 39 40 41 34 15 Salmonella s t r a i n TA 100 Sample Extract concentration (ul/ml) 8.33 4.17 2.08 1.04 0.52 0.0 B l 39, 39" 30, 32 30, 29 26, 24 14, 27 16, 15 B2 47 44 31 24 18 21 VI 47, 49 38, 41 33, 43 18, 21 16, 20 23, 13 V2 38 35 33 23 18 14 V3 39 27 32 25 19 11 HI 50 40 33 29 18 12 H2 48 45 43 26 15 11 H3 46 29 31 24 17 11 Baseline d i e t Vegetarian d i e t High meat di e t Experiment repeated to test f o r consistency of r e s u l t s 158 TABLE L.2 Number of Fluctuation Test Tubes Containing Revertant Bacteria A f t e r 5 Days, Subject Number 2 Salmonella s t r a i n TA 98 Sample Extract concentration (yl/ml) 8.33 4.17 2.08 1.04 0.52 0.0 B l 1 34 29 27 25 30 20 B2 40 28 28 22 21 19 VI 2 42 43 36 35 24 16 V2 37 35 24 39 40 19 V3 40 38 27 31 42 22 HI 3 50 34 30 37 33 17 H2 50 39 42 39 42 22 . H3 50 41 32 36 41 17 Salmonella s t r a i n TA 100 Sample Extract concentration (yi/mi) 8.33 4.17 2.08 1.04 0.52 0.0 B l 43 40 34 18 13 22 B2 39 25 21 17 16 20 VI 49 41 40 28 29 19 V2 35 30 31 31 34 20 V3 47 35 28 21 15 22 HI 45 37 43 33 32 19 H2 44 46 39 30 23 21 H3 50 41 43 27 23 20 Baseline d i e t Vegetarian d i e t 3 High meat d i e t 159 TABLE L.3 Number of Fluctuation Test Tubes Containing Revertant Bacteria A f t e r 5 Days, Subject Number 3 Salmonella s t r a i n TA 98 Sample Extract concentration Cyl /mi ) 8.33 4.17 2.08 1.04 0.52 0.0 B l 1 35 43 38 37 32 18 B2 27 40 36 37 36 17 V I 2 50 40 39 33 35 17 V2 50 46 32 38 34 22 V3 50 42 44 31 34 19 HI 3 50 40 40 37 34 11 H2 50 46 39 42 40 17 H3 50 50 37 37 32 20 Salmonella s t r a i n TA 100 Sample Extract concentration (yl/ml) 8.33 4.17 2.08 1.04 0.52 0.0 Bl 46 45 47 37 25 24 B2 46 33 29 41 31 14 VI 35 34 41 34 20 22 V2 50 46 39 32 19 13 V3 50 39 27 33 29 16 HI 50 41 41 32 14 20 H2 50 46 41 39 29 18 H3 50 42 39 39 26 21 Baseline d i e t 2 Vegetarian d i e t High meat di e t TABLE L.4 Number of Fluctuation Test Tubes Containing Revertant Bacteria A f t e r 5 Days, Subject Number 4 Salmonella s t r a i n TA 98 Sample Extract concentration (yl/ml) 8.33 4.17 2.08 1.04 0.52 0.0 B l 1 40 29 26 30 20 14 B2 33 26 22 31 24 16 V I 2 48 30 32 26 21 15 V2 50 47 32 28 24 17 V3 40 34 33 23 32 17 HI 3 45 36 34 31 12 15 H2 50 40 36 32 34 16 H3 50 49 33 29 29 14 Salmonella s t r a i n TA 100 Sample Extract concentration (yl/ml) 8.33 4.17 2.08 1.04 0.52 0.0 Bl 27 23 38 40 17 17 B2 23 24 21 19 22 14 VI 49 32 37 26 20 19 V2 43 44 36 32 20 18 V3 47 43 34 29 18 16 HI 50 40 23 19 14 13 H2 47 29 32 20 15 13 H3 35 42 21 24 24 10 Baseline d i e t 2Vegetarian d i e t 3High meat di e t TABLE L.5 Number of Fluctuation Test Tubes Containing Revertant Bacteria After 5 Days, Subject Number 5 Salmonella s t r a i n TA 98 Sample Extract concentration (ul/ml) 8.33 4.17 2.08 1.04 0.52 0.0 B l 1 38 39 28 24 24 16 B2 35 34 31 25 19 14 V I 2 26 22 32 29 21 19 V2 25 32 26 33 26 21 V3 27 25 25 32 30 9 HI 3 26 25 25 17 1 20 15 H2 34 24 20 27 25 18 H3 35 31 22 23 21 15 Salmonella s t r a i n TA 100 Sample Extract concentration CP1/ml) 8.33 4.17 2.08 1.04 0.52 0.0 Bl 48, 451* 41, 41 43, 35 25 23 18, 21 B2 47, 48 38, 38 29 18, 27 22, 23 13, 21 VI 35, 38 35, 34 22, 24 26 16 14, 22 V2 39 42 40 21 17 21 V3 38 43 36 24 20 20 HI 43 42 47 35 29 21 H2 37 41 33 20 22 23 H3 42 26 39 26 21 20 1Baseline d i e t 2Vegetarian d i e t 3High meat di e t it Experiment repeated to test f o r consistency of results. TABLE L.6 Number of F l u c t u a t i o n Test Tubes Containing Revertant B a c t e r i a A f t e r 5 Days, Subject Number 6 Salmonella s t r a i n TA 98 Extract concentration ( ul/ml) Sample 8.33 4.17 2.08 1.04 0.52 0.0 B l 1 45, 42 " 35, 39 27, 31 23, 26 26, 24 16, 13 B2 50 41, 38 33, 30 33, 29 23, 24 16, 13 VI 2 32 30 32 24 19 13 V2 46 27 30 22 17 20 V3 37 31 35 26 26 19 HI 3 41 29 30 21 30 14 H2 46 29 29 25 24 16 H3 49 27 27 29 24 14 Salmonella s t r a i n TA 100 Sample Extract concentration (Jjl/ml) 8.33 4.17 2.08 1.04 0.52 0.0 B l 49 41 47 38 20 20 B2 50 36 43 31 29 19 VI 43 48 47 31 21 14 V2 40 27 27 20 15 13 V3 36 25 29 22 24 18 HI 50 44 34 39 23 20 H2 35 27 21 23 15 21 H3 50 47 35 29 19 18 Baseline d i e t 2Vegetarian d i e t 3High meat d i e t "*Experiment repeated to t e s t f o r consistency of r e s u l t s 

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