Xenobiotic Metabolism: Nutritional Effects 9780841209121, 9780841211087, 0-8412-0912-X

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Publication Date: May 6, 1985 | doi: 10.1021/bk-1985-0277.fw001

Xenobiotic Metabolism: Nutritional Effects

Publication Date: May 6, 1985 | doi: 10.1021/bk-1985-0277.fw001

ACS

SYMPOSIUM

SERIES

Xenobiotic Metabolism: Nutritional Effects John W. Finley, EDITOR Publication Date: May 6, 1985 | doi: 10.1021/bk-1985-0277.fw001

Nabisco Brands, Inc.

Daniel E. Schwass, EDITOR Oregon Health Sciences University

Based on a symposium sponsored by the Division of Agricultural and Food Chemistry at the 187th Meeting of the American Chemical Society, St. Louis, Missouri, April 8-13, 1984

American Chemical Society, Washington, D.C. 1985

277

Library of Congress Cataloging in Publication Data Xenobiotic metabolism. (ACS symposium series, ISSN 0097-6156; 277)

Publication Date: May 6, 1985 | doi: 10.1021/bk-1985-0277.fw001

"Based on a symposium sponsored by the Division of Agricultural and Food Chemistry at the 187th meeting of the American Chemical Society, St. Louis, Missouri, April 8-13, 1984." Includes bibliographies and indexes. 1. Xenobiotics—Metabolism—Nutritional aspects— Congresses. I. Finley, John W., 1942.II. Schwass, Daniel E., 1951. III. American Chemical Society. Division of Agricultural and Food Chemistry. IV. American Chemical Society. Meeting (187th: 1984: St. Louis, Mo.) V. Series. QP529.X46 1985 ISBN 0-8412-0912-X

612'.39

85-6191

Copyright © 1985 American Chemical Society All Rights Reserved. The appearance of the code at the bottom of the first page of each chapter in this volume indicates the copyright owner's consent that reprographic copies of the chapter may be made for personal or internal use or for the personal or internal use of specific clients. This consent is given on the condition, however, that the copier pay the stated per copy fee through the Copyright Clearance Center, Inc., 21 Congress Street, Salem, M A 01970, for copying beyond that permitted by Sections 107 or 108 of the U.S. Copyright Law. This consent does not extend to copying or transmission by any means—graphic or electronic—for any other purpose, such as for general distribution, for advertising or promotional purposes, for creating a new collective work, for resale, or for information storage and retrieval systems. The copying fee for each chapter is indicated in the code at the bottom of the first page of the chapter. The citation of trade names and/or names of manufacturers in this publication is not to be construed as an endorsement or as approval by ACS of the commercial products or services referenced herein; nor should the mere reference herein to any drawing, specification, chemical process, or other data be regarded as a license or as a conveyance of any right or permission, to the holder, reader, or any other person or corporation, to manufacture, reproduce, use, or sell any patented invention or copyrighted work that may in any way be related thereto. Registered names, trademarks, etc., used in this publication, even without specific indication thereof, are not to be considered unprotected by law.

ACS Symposium Series Publication Date: May 6, 1985 | doi: 10.1021/bk-1985-0277.fw001

M . Joan Comstock, Series Editor Advisory Board Robert Baker U.S. Geological Survey Martin L . Gorbaty Exxon Research and Engineering Co.

Robert Ory USDA, Southern Regional Research Center Geoffrey D. Parfitt Carnegie-Mellon University

Roland F. Hirsch U.S. Department of Energy

James C. Randall Phillips Petroleum Company

Herbert D. Kaesz University of California—Los Angeles

Charles N . Satterfield Massachusetts Institute of Technology

Rudolph J. Marcus Office of Naval Research

W. D. Shults Oak Ridge National Laboratory

Vincent D. McGinniss Battelle Columbus Laboratories

Charles S. Tuesday General Motors Research Laboratory

Donald E . Moreland USDA, Agricultural Research Service

Douglas B. Walters National Institute of Environmental Health

W. H . Norton J. T. Baker Chemical Company

C. Grant Willson IBM Research Department

FOREWORD

Publication Date: May 6, 1985 | doi: 10.1021/bk-1985-0277.fw001

The ACS SYMPOSIUM SERIES was founded in 1974 to provide a

medium for publishing symposia quickly in book form. The format of the Series parallels that of the continuing ADVANCES IN CHEMISTRY SERIES except that, in order to save time, the papers are not typeset but are reproduced as they are submitted by the authors in camera-ready form. Papers are reviewed under the supervision of the Editors with the assistance of the Series Advisory Board and are selected to maintain the integrity of the symposia; however, verbatim reproductions of previously published papers are not accepted. Both reviews and reports of research are acceptable, because symposia may embrace both types of presentation.

Publication Date: May 6, 1985 | doi: 10.1021/bk-1985-0277.pr001

PREFACE C)UR ENVIRONMENT exposes us daily to a wide variety of xenobiotics: in our food, in the air we breathe, or as a result of industrial exposure and toxic wastes. However, despite this exposure, most of us are living long, healthy lives. Certainly individual variation could account for some of the variability in resistance to disease, but other factors are undoubtedly involved. According to a growing body of evidence, diet may be extremely important in increasing resistance to chronic disease. One is tempted to speculate, or hope, that improved dietary habits could improve individual resistance to chemically induced chronic disease. In this volume, we have focused on how xenobiotics are metabolized in higher animals and how this metabolism is mediated by the nutritional status of the target animal. Emphasis has been placed on the toxic, mutagenic, carcinogenic and potentially mutagenic or carcinogenic compounds. The topic is a natural follow-up and expansion of "Xenobiotics in Foods and Feeds" (ACS Symposium Series No. 234, Finley and Schwass, Eds.) in which sources of xenobiotics were identified and discussed at length. The authors in this volume discuss how and why these xenobiotics are toxic and how nutritional intervention can mediate some of the toxicities. Current nutritional awareness in the western world is probably unparalleled in the history of man. One need only look in health food stores and supermarkets to see the results of this awareness. Many food companies now place major emphasis on the natural, pure, low-calorie, additive-free, health- and fitness-oriented ingredients in their products. Nutritionists have established requirements for normal individuals, and additional data are being acquired rapidly on diets designed especially for individuals who experience high stress due either to illness or life style. This volume presents a state-of-the-art assessment of how diet can intervene and aid in the prevention of chronic disease. The editors hope this effort will stimulate further research in this important area of food biochemistry and nutrition. The authors wish to express their sincere gratitude to Miles Laboratories, Stroh Brewing Co., Nabisco Brands, Cutter Laboratories, Best Foods, General Mills, Inc., Warner Jenkinson, McCormick and Co., H. J. Heinz, and Lipton, Inc. for their generous support in helping many of the authors attend the symposium upon which this volume is based. J O H N W. FINLEY

D A N I E L E. SCHWASS

Nabisco Brands, Inc. Fair Lawn, New Jersey

Oregon Health Sciences University Portland, Oregon

September 21, 1984 ix

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The editors would like to dedicate this volume to the memory of Morris N. Joselow whose untimely death in 1983 was a loss to all, particularly those in the area of preventative medicine. Dr. Joselow was Professor of Preventative Medicine and Community Health at the College of Medicine and Dentistry in Newark, New Jersey. One of his last published works, "Systematic Toxicity Testing for Xenobiotics in Foods," appeared in "Xenobiotics in Foods and Feeds" (ACS Symposium Series No. 234). In addition to his other duties, he was organizer and principal lecturer in the American Chemical Society Toxicology Short Course. Dr. Joselow's research interests included environmental sciences, industrial hygiene and safety, toxicology, trace metals, and biochemical monitoring. During his career he published more than 100 papers. Dr. Joselow will be missed as a friend, a coworker, and a scientist.

1 Overview: The Influence of Nutrition on Xenobiotic Metabolism D. E. SCHWASS1,3 and J. W. FINLEY2 U.S. Department of Agriculture, Western Regional Research Center, Berkeley, CA 94610 Nabisco Brands, Inc., Fair Lawn,NJ07410

1 2

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In the course of living in the modern world it is inevitable that man and animals will be exposed to compounds in the environment which are not essential for life or even "normal" from the standpoint of the evolution of the species. The term "xenobiotic" (from the Greek "xenos" and "bios", meaning stranger to life) was coined by Mason, et al (1) to describe the myriad of compounds including carbohydrates, lipids, proteins, alkaloids, natural and synthetic drugs, flavorings, pigments, preservatives, polycyclic hydrocarbons, flavonoids, terpenoids, etc., which may enter the organism as non-essential or non-functional materials. The assumption inherent in the use of the term is that one is speaking relative to an organism of reference. For example, the drug quinine is a xenobiotic relative to man but not to the South American tree, Chinchona officinalis, in which quinine is a major constituent of the bark. Xenobiotics which enter the biosphere of the organism are not necessarily toxic. In fact, based on the Mason definition, non-essential amino acids could be referred to as xenobiotics. For the purpose of this symposium, however, xenobiotic does not include non-essential nutrients which occur in the diet, but will be restricted to environmental compounds which are acutely toxic, potentially toxic requiring activation, or which exhibit long term effects, such as mutagens, carcinogens or teratogens. In general, the discussions in this symposium are relative to man and/or animals. It is important to remember that xenobiotics range from the inocuous (i.e. vanillin) through the chronically toxic (i.e. ethanol) to the acutely toxic (i.e. curare). Some xenobiotics, although not toxic in and of themselves, are metabolically converted to toxic substances. The metabolic conversion of xenobiotics to toxic substances can be dramatically influenced by the nutritional status of the organism. Smoking, drugs, industrial chemicals and foods, represent the major sources of exposure to xenobiotics for modern man. Because diet furnishes the most variable and continuous array of xenobiotic exposure, the emphasis of this symposium is the 3

Current address: Oregon Health Sciences University, Portland, OR 97201

0097-6156/85/0277-0001 $06.00/0 © 1985 American Chemical Society

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XENOBIOTIC METABOLISM: NUTRITIONAL EFFECTS

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i n f l u e n c e of food-borne x e n o b i o t i c s and how d i e t can mediate the m e t a b o l i s m of t h e s e compounds. Food borne x e n o b i o t i c s c a n be endogenous t o the f o o d s t u f f ( f l a v i n o i d s i n t e a ) , can r e s u l t from p r o c e s s i n g and s t o r a g e ( l i p i d o x i d a t i o n o r non-enzymatic browning) or can be the r e s u l t of d e l i b e r a t e a d d i t i o n ( t h e antioxidant, butylated hydroxyanisole). Exposure t o x e n o b i o t i c s i s i n e v i t a b l e and a l t h o u g h the degree of exposure can be c o n t r o l l e d , i t i s i m p o s s i b l e t o p r e v e n t exposure a l t o g e t h e r . F o r t u n a t e l y , the h e a l t h y , w e l l - n o u r i s h e d i n d i v i d u a l o r a n i m a l under normal c i r c u m s t a n c e s can r e s i s t l o n g term e f f e c t s of many of t h e s e compounds by m e t a b o l i z i n g them and e x c r e t i n g them as m e t a b o l i t e s o r c o n j u g a t e s of m e t a b o l i t e s . A major s o u r c e of exposure to x e n o b i o t i c s i s tobacco smoke. C i g a r e t t e smoke c o n s i s t s of a l a r g e v a r i e t y o f compounds i n c l u d i n g o x i d a n t s , f r e e r a d i c a l s , b e n z o - ( a ) - p y r e n e and c a r b o n monoxide. Long term exposure t o c i g a r e t t e smoke e n t a i l s exposure t o both a c u t e l y t o x i c m a t e r i a l s and c h r o n i c a l l y t o x i c m a t e r i a l s . We a r e a l l exposed t o smoke i n v a r i o u s degrees b o t h from t o b a c c o and from the environment through the b u r n i n g of f u e l s and from c o o k i n g . Exposure t o i n d u s t r i a l s o u r c e s of x e n o b i o t i c s c a n come v i a the a i r o r through exposure and a b s o r p t i o n by the s k i n . A l t h o u g h major i n d u s t r i a l exposure r e c e i v e s much p u b l i c i t y when i t o c c u r s . T h i s t y p e of event u s u a l l y a c c o u n t s f o r exposure t o r e l a t i v e l y few compounds over a p r o l o n g e d p e r i o d of t i m e . F o r t u n a t e l y , c h e m i c a l companies have made g r e a t s t r i d e s i n r e d u c i n g the i n c i d e n c e of such exposure, a l t h o u g h i t i s l i k e l y t h a t some exposure w i l l c o n t i n u e t o o c c u r e i t h e r through a c c i d e n t s o r l a c k of knowledge. I n d u s t r i a l p o l l u t i o n i n the form of t o x i c waste s h o u l d be reduced s i g n i f i c a n t l y i n the next s e v e r a l y e a r s as e f f o r t s c o n t i n u e t o c o r r e c t t h i s problem. Exposure from t o x i c waste dumps i s l i k e l y t o c o n t i n u e but a t lower l e v e l s . P h a r m a c e u t i c a l drugs c a n be c o n s i d e r e d x e n o b i o t i c s and i n the w e s t e r n w o r l d an i n d i v i d u a l might expect t o be exposed s i g n i f i c a n t l y t o t w o - t o - t h r e e dozen compounds i n a l i f e t i m e under normal c i r c u m s t a n c e s . F r e q u e n t l y t h e s e exposures a r e over r e l a t i v e l y short p e r i o d s of time. Food r e p r e s e n t s a l a r g e and c o n t i n u o u s exposure t o a v a s t a r r a y of x e n o b i o t i c s . X e n o b i o t i c s from f o o d c a n range from t h e inocuous to the e x t r e m e l y dangerous. P l a n t s f r e q u e n t l y produce a v a r i e t y of x e n o b i o t i c s which s u b s e q u e n t l y a r e consumed by man o r animals. I n a d d i t i o n , d u r i n g s t o r a g e and p r o c e s s i n g of f o o d s , ( i n c l u d i n g home c o o k i n g ) , x e n o b i o t i c s c a n be produced. A recent symposium ( F i n l e y and Schwass) (2) reviewed many of t h e s e s o u r c e s of x e n o b i o t i c s i n t h e d i e t . H i s t o r i c a l l y , the impact of d i e t on h e a l t h has been a c o n c e r n of man. I n r e c e n t y e a r s we have seen g r e a t e r emphasis on how n u t r i t i o n r e l a t e s t o h e a l t h and the p r e v e n t i o n of c h r o n i c a f f l i c t i o n s such as c o r o n a r y h e a r t d i s e a s e , h y p e r t e n s i o n , o b e s i t y , and c a n c e r . C o n s e q u e n t l y , s e v e r a l h e a l t h o r g a n i z a t i o n s have proposed g u i d e l i n e s t o promote b e t t e r h e a l t h and reduce r i s k

1.

SCHWASS AND FINLEY

Overview

of c h r o n i c d i s e a s e *

The N a t i o n a l R e s e a r c h C o u n c i l

3

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(3) p u b l i s h e d an e x t e n s i v e study and g u i d e l i n e s r e g a r d i n g n u t r i t i o n a l means t o reduce t h e r i s k of c a n c e r . I n t h i s volume, Palmer (^) d i s c u s s e s t h e s e g u i d e l i n e s i n terms o f c u r r e n t e v i d e n c e . One o f the problem a r e a s d i s c u s s e d i s d i e t a r y f a t and i t s r e l a t i o n s h i p t o c a n c e r as w e l l as p r e v i o u s l y e s t a b l i s h e d r e l a t i o n s h i p s t o c o r o n a r y h e a r t d i s e a s e and o b e s i t y . Throughout t h i s symposium one o b s e r v e s a common t h r e a d i n many of t h e p a p e r s : the many problems a s s o c i a t e d w i t h l i p i d s i n the d i e t . I t i s important t o n o t e however, t h a t the problems may not be due s i m p l y t o f a t but more l i k e l y t o o x i d i z e d l i p i d products. P r y o r (2) r e v i e w s the mechanisms o f l i p i d o x i d a t i o n and d i s c u s s e s t h e c h e m i c a l b a s i s f o r a r e l a t i o n s h i p between l i p i d o x i d a t i o n and c h r o n i c d i s e a s e . I t would seem from t h i s and o t h e r evidence (D (§) t h a t p e r o x i d i z i n g l i p i d s c o u l d be a major d i e t a r y c o n c e r n i n the development o f c e r t a i n t y p e s o f c a n c e r s . I f one c o n s i d e r s t h e e a r l y s t a g e s o f tumor development t o be i n i t i a t i o n f o l l o w e d by promotion, one c o u l d s p e c u l a t e a number o f roles for peroxidizing l i p i d s . I n i t i a t i o n f o r the purpose o f t h i s d i s c u s s i o n w i l l be c o n s i d e r e d t h e i n i t i a l c h e m i c a l change i n the DNA o f a c e l l which has the p o t e n t i a l t o l e a d t o t h e development o f a tumor. I n t h e p r o m o t i o n s t a g e , t h e damaged c e l l b e g i n s t o m u l t i p l y as a r e s u l t o f c h e m i c a l i n s u l t and tumor development p r o c e e d s . The i n i t i a l damage o r i n i t i a t i o n c a n be caused by a v a r i e t y o f compounds, many o f which a r e used a s model compounds f o r t h e study o f c a r c i n o g e n e s i s . One might s p e c u l a t e t h a t these compounds ( i . e . , DMBA, b e n z o - ( a ) - p y r e n e ) c o u l d a c t a s i n i t i a t o r s and t h e o x i d i z i n g l i p i d s as promoters. Indeed, much of t h e e v i d e n c e i n the p r e s e n t symposium w i l l support t h i s speculation. I f one then c o n s i d e r s t h e p r o t e c t i v e r o l e a g a i n s t tumor development o f r e t e n o i d s , BHT, a s c o r b i c a c i d and t o c o p h e r o l , a l l o f which a r e a n t i o x i d a n t s , t h e argument i s s t r e n g t h e n e d (Chow, (1) (JO: K i n g , ; Newberne. ). The opinions about implications for public policy do not necessarily reflect those of the Food and Nutrition Board or the National Research Council. Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9.

American Cancer Society. "Nutrition and Cancer: Cause and Prevention"; American Cancer Society: New York, 1984; p. 15. American Heart Association, Committee on Nutrition. Circulation 1982, 65(4), 839A-854A. American Medical Association, Council on Scientific Affairs. J. Am. Med. Assoc. 1979, 242, 2335-8. "Healthy People: The Surgeon General's Report on Health Promotion and Disease Prevention," U.S. Department of Health, Education, and Welfare, 1979, p. 177. "Toward Healthful Diets," National Academy of Sciences, 1980. "Diet, Nutrition, and Cancer," National Academy of Sciences, 1982, p. 496. "Dietary Goals for the United States, Second Edition," U.S. Senate Select Subcommittee on Nutrition and Human Needs, 1977. "Nutrition and Your Health—Dietary Guidelines for Americans," U.S. Department of Agriculture and Department of Health, Education, and Welfare, 1980, p. 20. "Statement on Diet, Nutrition, and Cancer," by Upton, A. C., Senate Committee on Agriculture, Nutrition, and Forestry, October 2, 1979.

PALMER

10. 11. 12. 13.

14. 15.

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16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27.

28. 29. 30. 31. 32. 33. 34.

Dietary Guidelines to Reduce Cancer Risks

Doll, R.; Peto, R. J. Natl. Can. Inst. 1981, 66, 1192-1308. Stamler, J. Biometrics 1982, 37 (Suppl.), 95-114. Hiatt, H. H.; Watson, J. D.; Winsten, J. A., Eds.; ORIGINS OF HUMAN CANCER, 3 volumes, Cold Spring Harbor Laboratory: Cold Spring Harbor, New York, 1977. Doll, R. In "Introduction"; Hiatt, H. H.; Watson, J. D.; Winsten, J. A., Eds.; ORIGINS OF HUMAN CANCER, 3 volumes, Cold Spring Harbor Laboratory: Cold Spring Harbor, New York, 1977. Higginson, J.; Muir, C. S. J. Natl. Can. Inst. 1979, 63, 1291-8. Wynder, E. L.; Gori, G. B. J. Natl. Can. Inst. 1977, 58, 825-32. Reddy, B. S.; Cohen, L. A.; McCoy, G. D.; Hill, P.; Weisburger, J. H.; Wynder, E. L. Adv. Can. Res. 1980, 32, 237-345. Palmer, S. Cancer Res. (Suppl.) 1983, 43, 25095-25145. Ahrens, E. H., Connor, W. E. Am. J. Clin. Nutr. 1979, 36, 2621-2748. "Diet, Nutrition, and Cancer: Directions for Research," National Academy of Sciences, 1983, p. 73. Ames, B. N. Science 1983, 221(4617), 1256-64. McNutt, K. Nutr. Rev. 1980, 38, 353-60. Palmer, S.; Bakshi, K. J. Natl. Can. Inst. 1983, 70, 1153-70. Lipid Research Clinics Program. J. Am. Med. Assoc. 1984, 251(3), 351-64. "Diet and Exercise," Stockholm National Board of Health and Welfare, 1972, p. 36. "Swedish Nutrition Recommendations," Swedish National Food Administration, 1981, p. 11. "On Norwegian Food and Nutrition Policy," Royal Norwegian Ministry of Agriculture, 1975. "Recommendations for Prevention Programs in Relation to Nutrition and Cardiovascular Disease," Bureau of Nutritional Sciences, Health Protection Branch, Canadian Department of National Health and Welfare, 1977. Molitor, G. T. "National Nutrition Goals--How Far Have We Gone?;" Chou, M.; Harmon, D. P., Eds.; CRITICAL FOOD ISSUES OF THE EIGHTEES, Pergamon Press: New York. "Nutrition Policy Statements," Commonwealth Department of Health and National Health and Medical Research Council, 1983, p. 39. Langford, W. A. Food and Nutr. Notes and Rev. 1979, 36, 100-03. Walker, W. J. 1983. N. Engl. J. Med. 1983, 308(11), 64951. Rose, G. Br. Med. J. 1981, 282, 1847-51. "Recommended Dietary Allowances," National Academy of Sciences, 1980, 9th ed. Anonymous. Lancet 1983, 2(8352), 719-21.

RECEIVED

August 17,

1984

3 The Inhibition and Promotion of Cancers by Folic Acid, Vitamin B , and Their Antagonists 12

VICTOR HERBERT

Publication Date: May 6, 1985 | doi: 10.1021/bk-1985-0277.ch003

Hahnemann University, Philadelphia, PA 19102 For the past two years, our group has been collaborating with that of Dr. Ludwik Gross in the area of these studies, attempting to "switch off" the oncogene for guinea pig leukemia/lymphoma (1). The concept involved is that DNA methylation, and specifically methylation of cytosine in higher eukaryotes can directly suppress gene expression. This concept has been elaborated in several reviews, of which the most recent is by Eick et al. (2) in Analytical Biochemistry 135:165-171, 1983. The first dramatic presentation of possible clinical value of being able to demethylate a gene was the study by Heller and his associates in Chicago suggesting that they could "switch on" the fetal hemoglobin gene using 5-azacytidine, presumably by hypomethylating the fetal globin gene. They collaborated with Ley et al. in a study strongly suggesting that they could, in fact, switch on fetal hemoglobin synthesis with 5-azacytidine (3). This was confirmed by Charache and Dover and their associates at Johns Hopkins University (4) but Nathan and Lethvin and their associates showed that two other S-phase specific cytotoxic agents, hydroxyurea and cytosine arabinoside, could also increase fetal hemoglobin synthesis, Stanatiannopoulos and Poppianopoulou found that cytosine arabinoside can produce identical response in baboons to 5-azacytidine, and Nathan was quoted as concluding that the three drugs "probably act in the same way. Methylation has nothing to do with it" (5). However, W. French Anderson and his associates were able to show directly in the cell culture system that 5-azacytidine does in fact selectively hypomethylate fetal globin genes, but that other genes, including an oncogene, are remethylated shortly after losing their methyl groups. These superficially divergent results can be reconciled by the concept that hypomethylation causes the persistent hemoglobin, whereas other mechanisms produce the acute increased production of fetal hemoglobin which occurs after 5-azacytidine (or hydroxyurea or cytosine arabinoside (5). Similarly, different acute and persistent effects may explain 0097-6156/85/0277-0031 $06.00/0 © 1985 American Chemical Society

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why the same antifolate, methotrexate, which can shut down a lympho-proliferative malignancy, may result years later in the development of a second malignancy (1). One can speculate that an acute toxic effect kills the tumor but that the same methotrexate which acutely toxically kills the tumor, in a long period of time will result in demethylation of an oncogene, which can then be expressed as second malignancy. Other evidence that demethylation can cause the expression of malignancies comes from studies by Feinberg and Vogelstein at Johns Hopkins (6) at the Institute Pasteur by Bourgeois and her associates, who found that glucocorticoids can cause expression of murine mammary tumor virus (MMTV) by glucocorticoid-induced methylation of long-terminal repeat sequences (7). Similarly, Proirier and his associates published a number of studies delineating the ability of methyl-deficient, amino acid-defined diets to produce liver tumors in rats treated both with and without initiating doses of diethylnitrosamine (8, 9). Their studies indicate that diethylmethyl deficiency markedly promotes liver carcinogenesis and exhibits complete carcinogenetic activity in this organ in the rat. Rogers and Newberne had shown that dietary methyl deficiency enhances the activities of a number of hepatocarcinogens, and Shinizuka and Lombardi have found that choline deficiency enhanced the hepatocarcinogenic activities of several agents. The concept that deficiency of folate or vitamin B-12, or any other cause of failure to methylate DNA and/or RNA can activate malignancy by hypomethylation or oncogenes, and that methylating oncogenes can inhibit malignancy by making them dormant, is similar to the concept of "relaxed control" of RNA synthesis. In the f50s, Mendel and Borek (10) had noted that when an organism autotrophic from methionine is deprived of methionine, it loses its ability to suppress synthesis of RNA, which is then synthesized more rapidly. It was speculated that deficiency of B-12 or folate could produce similar "relaxed control". It is possible that some forms of vitamin B-12 and of folic acid may act as inhibitors of methylation and other forms as promoters of methylation of RNA and DNA (1). Reduced forms of folic acid are metabolically active; oxidized forms may be antimetabolites (1). Hydroxocobalamin is metabolically active; cyanocobalamin can be a B-12 antimetabolite (1). Leuchtenberger et al. had reported that inositol Inhibited animal tumor growth but various B vitamins did not (11, 12). This requires reinvestigation to determine whether inositol can methylate oncogenes. Oxidized folate monoglutamate, which is not a metabolically active form of the vitamin, not only did not inhibit spontaneous breast cancer in mice but actually produced a more rapid growth of the primary tumors and a significant increase in lung metastases. This work requires repeating today, particularly from the point of view of whether metabolically

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HERBERT

Inhibition and

Promotion of Cancers

i n a c t i v e f o l a t e , t h a t i s , o x i d i z e d f o l a t e , can promote tumor development but m e t a b o l i c a l l y a c t i v e f o l a t e , p a r t i c u l a r l y the v e r y a c t i v e t r i g l u t a m a t e s , can promote m e t h y l a t i o n of oncogenes and t h e r e b y i n h i b i t t h e i r e x p r e s s i o n . F o r many y e a r s , a number of workers have been e x p l o r i n g the q u e s t i o n of whether one form of a v i t a m i n can be a growth promoter by a c t i n g as a coenzyme ( i . e . , a promoter of normal and tumor c e l l growth) w h i l e a n o t h e r form of the same v i t a m i n c a n a t t a c h t o the same apoenzyme or o t h e r l i g a n d ( s u c h as a v i t a m i n t r a n s p o r t i n g p r o t e i n ) and t h e n jam the machinery, j u s t as a key w i t h a t o o t h m i s s i n g can f i t i n t o a l o c k and t h e n not t u r n . The answer t o that question i s c l e a r l y yes. S l i g h t t o major d i f f e r e n c e s i n the same v i t a m i n s t r u c t u r e ( i . e . , a n a l o g u e s and congeners) b o t h e x i s t i n n a t u r e and a r e s y n t h e s i z a b l e ; some of them a r e a n t a g o n i s t s o r a n t i - v i t a m i n s which can be c r e a t e d from v i t a m i n s by o n l y s l i g h t l y warping t h e i r s t r u c t u r e ( 1 ) . F a r b e r et a l . (13) r e p o r t e d from H a r v a r d g i v i n g p t e r o y l t r i g l u t a m i c a c i d ( t e r o p t e r i n ) and p t e r o y l d i g l u t a m i c a c i d ( d i o p t e r i n ) , b o t h s y n t h e s i z e d by U. SubbaRow and h i s a s s o c i a t e s a t L e d e r l e L a b o r a t o r i e s , t o 90 p a t i e n t s w i t h v a r i o u s malignancies, noting that " i n general, adult p a t i e n t s experienced improvement i n energy, a p p e t i t e , sense of w e l l being...might be a s c r i b e d t o improved morale r e s u l t i n g from f r e q u e n t v i s i t s , more m e d i c a l a t t e n t i o n . . . " They a l s o r e p o r t e d i n c o n s t a n t temporary d e c r e a s e s i n s i z e of m e t a s t a s e s i n some tumors and d e g e n e r a t i o n and n e c r o s i s i n o t h e r s . The a p o c r y p h a l s t o r y i s t h a t Dr. F a r b e r was a l s o g i v i n g f o l i c a c i d ( t h e o x i d i z e d , s t a b l e p h a r m a c e u t i c a l form of the v i t a m i n ) t o c h i l d r e n with l y m p h o p r o l i f e r a t i v e malignancies (lymphocytic leukemia and lymphoma) u n t i l one of h i s r e s i d e n t s c o l l e c t e d s u f f i c i e n t d a t a t o suggest t h a t the c h i l d r e n r e c e i v i n g t h i s new v i t a m i n were d y i n g f a s t e r t h a n t h o s e c h i l d r e n not r e c e i v i n g i t . T h i s o b s e r v a t i o n a l l e g e d l y l e d Dr. F a r b e r t o ask the L e d e r l e p e o p l e t o c r e a t e a warped f o l i c a c i d m o l e c u l e which would i n t e r f e r e w i t h f o l a t e metabolism i n the m a l i g n a n t c e l l s , and t h i s was done by a d d i n g an NH2 group, t h e r e b y c r e a t i n g a m i n o p t e r i n . A second a l t e r a t i o n , m e t y h y l a t i o n i n the 1 0 - p o s i t i o n , c r e a t e d m e t h o t r e x a t e , s t i l l one of our most p o t e n t a n t i - c a n c e r a g e n t s , p a r t i c u l a r l y e f f e c t i v e against childhood lymphoproliferative d i s o r d e r s and t r o p h o b l a s t i c m a l i g n a n c i e s . There i s c o n s i d e r a b l e e v i d e n c e t h a t r a p i d l y growing n e o p l a s t i c t i s s u e consumes f o l a t e a t so r a p i d a r a t e t h a t f o l a t e d e f i c i e n c y m e g a l o b l a s t o s i s can o c c u r i n the h o s t c e l l s (14, 1 5 ) . There i s a l s o e v i d e n c e t h a t v i t a m i n B-12 d e f i c i e n c y may slow tumor growth, whether t h a t d e f i c i e n c y r e s u l t s f r o m i n a d e q u a t e a b s o r p t i o n o r e l e v a t e d l e v e l s i n serum of a v i t a m i n B-12 binder which does not d e l i v e r the v i t a m i n t o tumor t i s s u e ( 1 6 ) , but w i l l d e l i v e r i t t o the l i v e r i n a calcium-dependent f a s h i o n (14, 17-20). I n t e r e s t i n g l y , g r a n u l o c y t e s and l i v e r a r e a major s o u r c e

34

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of serum b i n d i n g p r o t e i n s f o r b o t h b i t a m i n B-12 and f o l i c a c i d (21), and m a l i g n a n c i e s o f g r a n u l o c y t e s and l i v e r may p a r t l y c o n t r o l themselves by r e l e a s i n g i n t o t h e serum l a r g e amounts o f b i n d e r s f o r v i t a m i n B-12 and f o l i c a c i d which b i n d t h o s e v i t a m i n s and t h e r e b y p r e v e n t d e l i v e r y t o , and nourishment o f , t h e tumor. O x i d i z e d f o l a t e i s not o n l y m e t a b o l i c a l l y dead buyt may even be n e u r o t o x i c . F o r example, a p a t i e n t w i t h e p i l e p s y who has not had a c o n v u l s i o n i n y e a r s because d i l a n t i n has produced complete c o n t r o l , c a n be thrown i n t o an immediate c o n v u l s i o n w i t h a megadose of f o l i c a c i d , because f o l i c a c i d and d i l a n t i n compete f o r a b s o r p t i o n a t the b r a i n c e l l s u r f a c e , and t o o much o x i d i z e d f o l i c a c i d w i l l b l o c k the a b i l i t y of the b r a i n c e l l t o t a k e up d i l a n t i n , s i m i l a r t o t h e c o m p e t i t i o n between d i l a n t i n and f o l i c a c i d f o r uptake by the g u t c e l l ( 2 2 ) . There appear t o be one-way t r a n s p o r t systems t o remove o x i d i z e d f o l a t e s from the nervous system (22, 23) and t o remove v i t a m i n B-12 analogues from t h e body v i a the b i l e ( 2 4 ) . Some of the B-12 analogues p r e s e n t i n m u l t i v i t a m i n / m i n e r a l p r e p a r a t i o n s may b l o c k mammalian c e l l metabolism (25) and s i n c e they b l o c k normal c e l l metabolism, p o s s i b l y may UTbck m a l i g n a n t c e l l metabolism. F o r a number of y e a r s , R u s s i a n workers have been f e e d i n g analogues of v i t a m i n B-12 t o normal and m a l i g n a n t c e l l s and showing t h a t t h e s e analogues w i l l knock out B-12 metabolism (25a). Do the B-12 analogues (which have now been found i n human serum, l i v e r , b i l e r e d c e l l s , and b r a i n ) (26) p l a y any r o l e i n the p r o m o t i o n o r i n h i b i t i o n of c a r c i n o g e n e s i s i n humans? Levels of analogue i n serum a r e e l e v a t e d i n some m a l i g n a n c i e s ( 1 ) . L e v e l s of m e t h y l a t e d bases i n u r i n e a r e e l e v a t e d i n some h e m a t o l o g i c m e l i g n a n c i e s ( J L ) . I n t h e same h e m a t o l o g i c a l m a l i g n a n c i e s i n which m e t h y l a t e d bases a r e e l e v a t e d i n the u r i n e , B-12 analogue i s e l e v a t e d i n t h e b l o o d serum ( 1 ) . We have r e c e n t l y found enormous q u a n t i t i e s of a n a l o g u e s i n human s t o o l , and have been s t u d y i n g whether t h e analogue i n human c o l o n b a c t e r i a i s the s o u r c e of t h e analogue i n human t i s s u e s ( 2 7 ) . I n p r e l i m i n a r y s t u d i e s , we found two l a r g e analogue peaks i n human b i l e and two s i m i l a r l a r g e analogue peaks i n human s t o o l . We a r e now a t t e m p t i n g t o f i n d out whether t h e s e peaks a r e t h e same analogue ( 2 7 ) . I f they a r e , t h e n the analogue i n b i l e would have come from t h e analogue i n s t o o l , because t h e q u a n t i t y o f analogue p r e s e n t i n f o o d i s t i n y compared t o the q u a n t i t y p r e s e n t i n human stool. Working w i t h Dr. Ludwik G r o s s i n our f i r s t attempts t o m e t h y l a t e oncogenes, we gave 5 - m e t h y l c y t i d i n e (5mC) t o g u i n e a p i g s i n whom was t r a n s p l a n t e d g u i n e a p i g leukemia/lymphoma ( 1 ) • The e x p e r i m e n t s o v e r a six-month p e r i o d were u n s u c c e s s f u l i n showing any d r a m a t i c i n h i b i t i o n , a l t h o u g h t h e r e was a non-statistically significant inhibition. We s u b s e q u e n t l y began g i v i n g 5 - i o d o c y t i d i n e (5IC) t o t h e s e g u i n e a p i g s , h a v i n g s w i t c h e d from 5MC because of the e v i d e n c e t h a t t h e m e t h y l group i s t a k e n

HERBERT

Inhibition and Promotion of Cancers

off on passage through the liver, resulting in cytidine alone being incorporated into the DNA and RNA of the oncogene. We though that perhaps iodine would not be removed from the cytidine as easily, and iodocytidine would be Incorporated intact into RNA, with the iodine perceived by the cell as if it were a methyl group, as is true for iododeoxyuridine being perceived by cells as if it were methyldeoxyuridine (i.e., thymidine) (1). These studies are not yet completed; preliminary results have been equivocal but teasing. Evidence that methylation can suppress normal and malignant gene expression, and demethylation can bring about expression, continues to build (28-30), although expression is not always related to state of methylation (31, 32).

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Literature Cited 1. Herbert, V. In "Nutritional Factors in the Induction and Maintenance of Malignancy"; Butterworth, C.E., and Hutchinson, M.L., Eds.; Academic Press: New York, 1983, pp. 273-287. 2. Eick, D., Fritz, H.-J., Doerfler, W.: Analyt. Biochem. 1983, 135, 165-171. 3. Ley, T.J., DeSimone, J., Noguchi, C.T., Turner, P. H., Schechter, A.N., Heller, P., Nienhuis, A.W., Blood 1983, 62, 370-380. 4. Charache, S., Dover, G., Smith, K., Talbot, C., Conover, C., Jr., Moyer, M., Boyer, S. Proc. Natl. Acad. Sci. 1983, 80, 4842-6. 5. Kolata, G. Science 1984, 223, 470-1. 6. Feinberg, A.P., Vogelstein, B. Nature 1983, 301, 89. 7. Mermod, J.-J., Bourgeois, S., Defer, N., Crepin, M. Proc. Natl. Acad. Sci. 1983, 80, 110-114. 8. Brown, J.D., Wilson, M.J., Poirier, L.A. Carcinogenesis 1983, 4, 173-177. 9. Mikol, Y.B., Hoover, K.L., Creasia, D., Poirier, L.A. Carcinogenesis 1983, 4. 10. Mandel, L.R., Borek, E. Biochem. Biophys. Res. Comm. 1961,

6, 138. 11. Leuchtenberger, C., Leuchtenberger, R., Laszlo, D., Lewisohn, R. Science 1945, 101, 46. 12. Leuchtenberger, C., Leuchtenberger, R. In: "Nutritional Factors in the Induction and Maintenance of Malignancy"; Butterworth, C.E., and Hutchinson, M.L., Eds.; Academic Press: New York, 1983, pp. 131-148. 13. Farber, S., Cutler, E.C., Hawkins, J.W., Harrison, J.H., Peirce, E.C., Lenz, G.G. Science 1947, 106, 619-621. 14. Herbert, V. "The Megaloblastic Anemias." Grune and Stratton, New York, 1959.

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15.

Chanaran, I. "The Megalblastic Anemias." Blackwell Scientific, St. Louis, 1979. 16. Corcino, J., Zalusky, R., Greenberg, M., Herbert, V. Brit. J. Haemat. 1971, 20, 511-520. 17. Herbert, V. J. Clin. Invest. 1958, 37, 646-650. 18. Herbert, V., Spaet, T.H. Amer. J. Physiol. 1958, 195, 194-196. 19. Allen, R.H. Prog. Hemat. 1975, 9, 57. 20. Beck, W.S. In: "B-12"; Dolphin, D., Ed. John Wiley and Sons: New York, 1982, pp 21. Herbert, V., Colman, N. In "Lithium Effects on Granulopoiesis and Immune Function"; Rossof, A.H., Robinson, W.A., Eds. Plenum Publishing: New York, 1980, pp. 61-78. 22. Colman, N., Herbert, V. In: "Biochemistry of Brain"; Kumar, S., Ed. Pergamon Press: New York, 1980, pp. 103, 125. 23. Poncz, M., Colman, N., Herbert, V., Schwartz, E., Cohen, A.R. J. Ped. 1981, 98, 76-79. 24. Kanazawa, S., Herbert, V. Trans. Assoc. Amer. Phys. 1984, 96, 336-344. 25. Kondo, H., Binder, M.J., Kolhouse, J.F., Smythe, W.R., Podell, E.R., Allen, R.H. J. Clin. Invest. 1982,k 70, 889-898. 25a. Myashcheva, N.W., Quadros, E.V., Matthews, D.M., Linnell, J.C. Biochim. Biophys. Acta 1979, 588, 81-88. 26. Kanazawa, S., Herbert, V. Clin. Res. 1982, 30, 540A. 27. Herbert, V., Drivas, G., Manusselis, C., Mackler, B., Eng, J., Schwarts, E. Trans. Assoc. Amer. Phys. 1984, 97. 28. Wilson, V.L., Jones, P.A. Cell 1983, 32, 239-246. 29. Christman, J.K., Mendolsohn, N., Herzog, D., Schneiderman, N. Cander Res. 1983, 43, 763-769. 30. Harrison, J.J., Anisowicz, A., Gadi, I.K., Raffeld, M., Sager, R. Proc. Natl. Acad. Sci. 1983, 80, 6606-66.0. 31. Gautsch, J.W., Wilson, M.C. Nature 1983, 301, 32-37. 32. Graessman, M., Graessmann, A., Wagner, H., Werner, E., Simon, D. Proc. Natl. Acad* Sci. 1983, 80, 6470-6474. RECEIVED January

23, 1985

4 The Influence of Fermentable Dietary Fiber on the Disposition and Toxicity of Xenobiotics J. DONALD DEBETHIZY1 and ROBIN S. GOLDSTEIN2 Rohm and Haas Company, Toxicology Department, Spring House, PA 19477 Smith, Kline, and French Laboratories, L-66, Philadelphia, PA 19101

1

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2

Fermentable dietary fiber may modulate chemical toxicity by altering the microfloral metabolism of xenobiotics. A series of studies were conducted to assess the influence of fermentable fiber on the toxicity of xenobiotics that require microfloral metabolism to express their toxicity. The hepatic macromolecular covalent binding of 2,6-dinitrotoluenederived radioactivity and nitrobenzene-induced methemoglobinemia were enhanced in rats fed pectin supplemented purified diets to levels comparable to rats fed cereal-based diets. The increased toxicity of these xenobiotics was associated with a 2- to 3-fold increase in the number of cecal anaerobic bacteria in rats fed the pectin diets. The number of cecal anaerobic bacteria in cereal-based diet-fed rats was similar to rats fed the purified diet supplemented with pectin. Following a single oral dose of Amaranth, the peak plasma concentration of naphthionic acid, a microfloral metabolite of Amaranth, was 5-fold higher in rats fed a pectin-supplemented, purified diet. These studies indicated that feeding diets containing fermentable fibers such as pectin can enhance the toxicity of nitroaromatics by increasing the number of cecal anaerobic bacteria that are required for the microfloral metabolism of these xenobiotics to proximate toxicants. Dietary fiber has been suggested to play a protective role against chemically-induced toxicity (1) and against colon cancer (2). However, the mechanism(s) by which dietary fiber modulates chemical toxicity or colon cancer has not been well studied. The fiber fraction of the diet is resistant to mammalian digestive enzymes and consequently dietary fiber is not absorbed from the small intestine (3). However, certain types of dietary fiber; specifically fermentable fibers, including the pectic substances and hemicelluloses, are readily digested by the intestinal microflora (4,5). Pectic 0097-6156/85/0277-O037$06.00/0 © 1985 American Chemical Society

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substances are a family of galacturonic acid polymers which are methoxylated t o varying degrees depending on the plant source (6). Hemicelluloses are primarily polymers of the pentose, xylose, with varying amounts of arabinose branching (7). Although hemicellulose constitutes the majority of the fermentable dietary f i b e r derived from most plant sources, i t has not been well studied because i t i s d i f f i c u l t to extract and i s o l a t e from plant c e l l walls (7). On the other hand, pectin, which i s the major component of pectic substances, i s e a s i l y isolated from apple and c i t r u s f r u i t s as a byproduct of the f r u i t j u i c e industry (8). Therefore, most studies on fermentable f i b e r have employed pectin as a model fermentable f i b e r . Animal feeds as well as human d i e t s vary considerably i n the type and quantity of dietary f i b e r . Wise and G i l b e r t (9) using modified detergent methods analyzed fourteen commercial rodent d i e t s and found that the t o t a l dietary f i b e r content varied from 8.3 to 22.4%. In f a c t , i t i s not unusual f o r commercially available cereal-based rodent diets to contain 20% dietary f i b e r on a dry weight basis (10). In general, the fermentable f i b e r s constitute more than h a l f of the t o t a l dietary f i b e r ; the remainder composed of the f i b e r s more resistant t o fermentation, such as c e l l u l o s e and l i g n i n (9). Thus, a s i g n i f i c a n t portion of rodent diets has the p o t e n t i a l to be fermented i n the i n t e s t i n a l t r a c t . One way i n which fermentable f i b e r could influence chemical t o x i c i t y i s by a l t e r i n g the m i c r o f l o r a l metabolism of xenobiotics. I t has been suggested that the fermentable components of dietary f i b e r s i g n i f i c a n t l y influence the i n t e s t i n a l m i c r o f l o r a l metabolism of xenobiotics by providing a p o t e n t i a l source of energy f o r microb i a l growth and a c t i v i t y (11). Using pectin as a model fermentable f i b e r , Bauer et a l . (12) demonstrated that there was a higher i n c i dence of dimethylhydrazine(DMH)-induced tumors of the colon i n Sprague-Dawley rats fed pectin-containing p u r i f i e d diets than i n rats fed a p u r i f i e d d i e t alone. These investigators speculated that pectin enhanced the metabolic a c t i v a t i o n of DMH as suggested by the concommitant elevation of m i c r o f l o r a l $-glucuronidase a c t i v i t y i n the pectin-fed animals. However, the r e l a t i o n s h i p between microfloral 3-glucuronidase a c t i v i t y and DMH-tumorigenicity has been questioned since there i s c o n f l i c t i n g evidence that hydrolysis of a glucuronide conjugate of DMH i s e s s e n t i a l f o r the a c t i v a t i o n of DMH to a proximate carcinogen (13). Amaranth Metabolism Our f i r s t i n d i c a t i o n that fermentable f i b e r could a l t e r m i c r o f l o r a l metabolism was based on studies assessing the influence of dietary f i b e r types on the d i s p o s i t i o n of model xenobiotics using pharmacok i n e t i c analysis (14). Amaranth was selected as a model xenobiotic for these studies because i t was absorbed only a f t e r reduction by gut microflora (15). In these studies adult, male Wistar rats were fed p u r i f i e d hydrated g e l a t i n d i e t s containing either no f i b e r or 15% c e l l u l o s e , l i g n i n , metamucil, or pectin (16). After 28 days on the d i e t the animals received a single o r a l dose of Amaranth (1 mmoleAg). Blood samples were c o l l e c t e d at various times and the plasma concentration of naphthionic acid (NA), the major m i c r o f l o r a l metabolite of

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39

Amaranth, was determined by high pressure l i q u i d chromatography (HPLC) (14). The fermentable f i b e r , pectin, elevated the peak plasma concent r a t i o n of NA 5-fold over the other dietary groups (Figure 1) (17). To determine i f the higher plasma concentration of NA was due to enhanced absorption of NA or increased m i c r o f l o r a l metabolism of Amaranth, the i n v i t r o metabolism of Amaranth by c e c a l contents from rats fed the various diets was examined i n a prelimary experiment. Cecal contents from animals fed the fiber-containing diets were incubated anaerobically with Amaranth and the amount of NA produced per gram of cecal contents determined by HPLC. Although the specif i c a c t i v i t y (on a per gram of cecal contents basis) of m i c r o f l o r a l Amaranth-azoreductase was lower i n pectin-fed animals, the t o t a l amount of NA produced per cecum was elevated 2-fold over rats fed the other diets (data not shown). These r e s u l t s suggested that feeding c i t r u s pectin to rats elevated the m i c r o f l o r a l metabolism of Amaranth r e s u l t i n g i n greater amounts of NA i n the plasma. These findings l e d us to believe that the capacity f o r m i c r o f l o r a l metabolism of xenobiotics i s enhanced by feeding pectin.

Dinitrotoluene Hepatotoxicity Based on the Amaranth studies, i t was hypothesized that those chemic a l s requiring m i c r o f l o r a l metabolism to express t h e i r t o x i c i t y may be more toxic to animals consuming fermentable f i b e r . 2,6-Dinitrotoluene (DNT) i s a hepatocarcinogen i n Fischer-344 rats (18) and i s genotoxic i n the i n v i v o / i n v i t r o hepatocyte DNA repair assay (19). The hepatic genotoxic i t y of DNT was found to be dependent upon the presence of gut microflora (20). Long and Rickert (21) demonstrated that DNT i s excreted i n the b i l e of male rats as the 2,6-dinitrobenzylalcohol glucuronide. This glucuronide conjugate i s hydrolyzed by m i c r o f l o r a l ^-glucuronidase, permitting DNT to undergo enterohepatic c i r c u l a t i o n . Evidence also indicated that similar to the hepatic genotoxicity of DNT, hepatic macromolecular covalent binding (CVB) was also dependent upon the presence of gut microflora (22). CVB therefore was used as an endpoint to test the hypothesis that pectin-containing diets could enhance the t o x i c i t y of xenobiotics by elevating m i c r o f l o r a l metabolism. In these experiments adult, male Fischer-344 rats were fed a p u r i f i e d d i e t , AIN-76A, containing 5 or 10% c i t r u s pectin replacing cornstarch or one of two c e r e a l based d i e t s , Purina Rodent Chow 5002 and NIH-07. After 28 days of dietary treatment rats were given a single o r a l dose of t r i t i a t e d DNT (10 or 75 mgAg). Twelve hours a f t e r dosing, animals were k i l l e d and CVB was determined by exhaust i v e extraction. The cecum was also excised from these animals and microflora characterized by anaerobic culture techniques (10). The CVB of DNT-derived r a d i o a c t i v i t y to hepatic macromolecules was independent of the d i e t at a dose of 10 mg DNTAg (Table I ) . However, at a dose of 75 mgAg, CVB was increased 40% and 90% by supplementing 5% and 10% pectin to the p u r i f i e d d i e t s , respectively. Livers of animals fed Purina 5002 and NIH-07 exhibited s i g n i f i c a n t l y greater CVB than animals fed the p u r i f i e d d i e t with or without pectin supplementation. CVB was increased approximately s i x f o l d when the dose of DNT was increased from 10 to 75 mgAg i n animals

XENOBIOTIC M E T A B O L I S M : NUTRITIONAL

7.00

EFFECTS

control col I u I ooo I Ign I n ao t u u o I I

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poet In

6

12

18 24 30 TIME

36 42 48 (hour.)

54

60

66 72

Figure 1. Concentration of naphthionic acid i n plasma from Wis tar rats given a single o r a l dose of Amaranth (1 mmole/kg) following feeding p u r i f i e d d i e t s containing no f i b e r (control) or 15% (w/w) c e l l u l o s e , l i g n i n , metamucil, or pectin f o r 30 days. Each point represents the mean of s i x r a t s .

4.

DEBETHIZY A N D GOLDSTEIN

Influence of Fermentable Dietary Fiber

41

fed NIH-07, Purina 5002, and p u r i f i e d d i e t plus 10% pectin, but increased only f o u r f o l d i n animals fed the p u r i f i e d d i e t or p u r i f i e d d i e t supplemented with 5% pectin.

Table I .

E f f e c t of Diet on the Hepatic Macromolecular Covalent Binding of DNT

Publication Date: May 6, 1985 | doi: 10.1021/bk-1985-0277.ch004

Hepatic Covalent Binding (nmol equivalents/g l i v e r ) A f t e r a DNT Dose of 10 mgAg 75 mgAg AIN-76A AIN-76A plus 5% pectin AIN-76A plus 10% pectin NIH-07 Purina 5002

1.03 1.21 1.11 1.40 1.47

+ + + + +

0.19 0.23 0.17 0.13 0.16

3.75 5.21 7.06 9.29 8.82

+ + + + +

0.22** 0.43 0.71

GSSG + 2H20

(1)

2GSH + R00H

->

GSSG + ROH

(2)

0097-6156/ 85/0277-0111$06.00/0 © 1985 American Chemical Society

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XENOBIOTIC M E T A B O L I S M : NUTRITIONAL EFFECTS

Selenium has been shown to be an unusually important dietary chemopreventative. In general, the dietary chemopreventative e f f e c t s have been demonstrated between 0.5 and 1.0 ppm. However, most animals have a dietary requirement of 0.1 to 0.2 ppm of selenium. The dietary requirement seems to be lower than the amount needed for the optimal chemopreventative effect and therefore may not be related to a minimal n u t r i t i o n a l requirement. On the other hand, one could also postulate that cancer i s a n u t r i t i o n a l l y related disease and that the real requirement i s around 0.5 ppm. In f i v e of s i x nondietary tumor-promotion experiments, sodium selenide s i g n i f i c a n t l y reduced the number of mice with tumors i n duced by 7,12-dimethyl-benzanthracene (DMBA)-croton o i l ( 1 ) . In these experiments, sodium selenide was applied concomitantly along with croton o i l to female Swiss albino mice i n i t i a t e d with DMBA. Riley has also observed a reduction in DMBA-phorbol ester c a r c i n o genesis by sodium selenide ( 2 ) . The effect of selenium-deficient and selenium-adequate diets on DMBA-croton o i l and benzopyrene skin carcinogenesis has also been studied. Supplemental dietary selenium i n h i b i t e d both types of carcinogenesis. Dietary selenium has also reduced carcinogen induced l i v e r carcinogenesis. Clayton and Baumann have reported that the i n c l u sion of 5 ppm of dietary selenium reduced the incidence of l i v e r tumors in rats induced by 3-methyl-4-dimethylaminoazobenzene (DAB) {3). S i m i l a r results were observed by G r i f f i n and Jacobs ( 4 J . Dzhioev has observed a marked reduction of l i v e r tumors induced by diethylnitrosamine (DEN) in the animals fed selenium diets ( 5 ) . Marked reduction of l i v e r tumors induced by acetyaminoflourene have been observed in rats fed dietary selenium {6) or given selenium in the drinking water {]_). Dietary selenium has also been shown to reduce the development of L-azaserine-induced preneoplastic abnormal acinar c e l l modules in male Wistar rats (8), the formation of a f l a t o x i n B induced gamma-glutamyltransferase~[GGTP) p o s i t i v e f o c i i n rat l i v e r (9) and the formation of GGTP p o s i t i v e f o c i induced by DEN (10). Fven though dietary selenium has an effect on skin and l i v e r carcinogenesis, even greater dietary effects have been observed on carcinogen and v i r a l l y induced breast cancer and carcinogen-induced colon cancer in animals. It may be of interest that breast and colon cancer have been shown to be enhanced by dietary fat in both man and animals. Perhaps t h i s enhancement i s due to an increase of fat peroxidation which can be reduced by antioxidants. Schrauzer and Ishmael have fed 2 ppm of selenium in the form of SeO^ in the drinking water for 15 months to v i r g i n C3H female mice wnich are e s p e c i a l l y susceptible to v i r a l l y induced spontaneous mammary t u mors induced by the B i t t n e r milk virus (lj_). The incidence of spontaneous mammary tumors was 82% in the untreated controls and 10% in the selenium treated mice. Thompson and Tagliaferro have observed that selenium supplemented diets have reduced the numbers of DMBA-induced mammary tumors per rat (12J. The fact that s e l e nium-supplemented diets have reduced both v i r a l l y and chemically induced cancer in animals indicates that both the v i r a l l y and chemi c a l l y induced carcinogenesis may have the same mechanism of induct i o n . Ip has studied the effect of selenium supplementation i n the 1

Publication Date: May 6, 1985 | doi: 10.1021/bk-1985-0277.ch009

9.

SHAMBERGER

Antioxidants and Malonaldehyde in Cancer

113

i n i t i a t i o n and promotion phase of DMBA-induced mammary carcinogenes i s in rats fed a high-fat diet (13). In t h i s experiment, rats were fed 5 ppm of sodium s e l e n i t e for various periods of time before and a f t e r treatment with DMBA. From t h i s experiment the f o l lowing conclusions were made: (1) both the i n i t i a t i o n and promotion phase of carcinogenesis can be i n h i b i t e d by selenium; (2) in order to achieve maximal i n h i b i t i o n of tumorigenesis, a continuous intake of selenium i s necessary; (3) the i n h i b i t o r y e f f e c t of selenium i n the early promotion phase i s probably r e v e r s i b l e ; (4) the u s e f u l ness of selenium i s decreased when i t i s given long a f t e r carcinogenic i n j u r y . Two types of epidemiological relationships have been found in two d i f f e r e n t populations. Both relationships were inverse to s e lenium b i o a v a i l a b i l i t y and p a r a l l e l e d the results from animal s t u d i e s . In one type of study, selenium b i o a v a i l a b i l i t y has been i n versely related to human cancer mortality in American c i t i e s and states (14-15). Schrauzer e t . a l . correlated the age-adjusted morta l i t y from cancer at 17 major body s i t e s with the apparent dietary selenium intakes estimated from food consumption data in 27 count r i e s (16). S i g n i f i c a n t inverse correlations were observed for cancers of the large i n t e s t i n e , rectum, prostate, breast, ovary, lung, and leukemia. In a d d i t i o n , weaker inverse associations were found for cancers of the pancreas, s k i n , and bladder. Vitamin E_ Vitamin E may prevent mouse skin tumorigenesis through i t s known antioxidant effect (1). Rats fed a diet containing large amounts of vitamin E had fewer mammary tumors induced by DMBA than did the controls {17). Shklar has observed that Syrian golden hamsters given oral vitamin E had fewer smaller buccal pouch cancers induced by DMBA (181. Konings and T r i e l i n g have observed an enhanced i n h i b i t i o n of~T H] thymidine incorporation into the DNA of vitamin Edepleted lymphosarcoma c e l l s (19). Weisburger e t . a l . have observed a greater incidence of stomach cancer in populations consuming low levels of vitamin E and other selected micronutrients. Vitamin C_ Vitamin C may prevent tumorigenesis through i t s antioxidant a c t i o n . Vitamin C i s water soluble and complements the antioxidant action of vitamin E which i s l i p i d s o l u b l e . When vitamin C was applied concomitantly with croton o i l to mouse skin previously treated with DMBA, the t o t a l number of mouse skin papillomas was reduced (20). S i m i l a r l y , Slaga and Bracken observed a decrease in the number of skin tumors induced by DMBA-phorbol carcinogenesis in mice treated with vitamin C (21). Tumor i n h i b i t i o n by ascorbic acid has also been observed on toad skin treated with DMBA (22). Schlegel e t . a l . have observed that vitamin C reduces u r o e p i t h e l i a l carcinoma i n mice and also suggested a s i m i l a r mechanism in humans (23). The tryptophan metabolite 3-hydroxyanthranilic acid (3-HOA) i s thought to be s t a b i l i z e d by ascorbic a c i d , thereby preventing carcinogenic i t y when 3-HOA i s implanted in the bladder. Vitamin C i s also known to prevent tumorigenesis through i t s a b i l i t y to block the 2IL

XENOBIOTIC M E T A B O L I S M : NUTRITIONAL E F F E C T S

114

v i t r o formation of N-nitroso compounds by the reaction between n i trous acid and oxytetracycline, morpholine, piperazine, N-methyla n i l i n e , methyl urea, and dimethyl amine. The amount of blocking depends on the compounds nitrosated and the experimental conditions (24). The species formed from nitrous acid responsible for the oxidation of ascorbic acid i s the same species a f f e c t i n g n i t r o s a t i o n of secondary amines (25). Between pH 1.5 and 5 . 0 , the n i t r o sation of secondary amines in the presence of ascorbic acid and the absence of oxygen can be summarized by the following two competit i v e p a r a l l e l second-order reactions:

Amine + N 0 2

^

3

Ascorbate + N 0 Publication Date: May 6, 1985 | doi: 10.1021/bk-1985-0277.ch009

2

3

k

2

->

nitrosamine + N0 " + H

->

dehydroascorbate + 2N0 + 2H 0

2

(3)

+

?

(4)

If k » k p then reaction (4) i s mostly complete before (3) starts. Large doses of vitamin C have been observed to protect rats from l i v e r tumors induced by aminopyrine and sodium n i t r i t e (26). This i n h i b i t i o n i s thought to r e s u l t , in p a r t , from blockage of in vivo n i t r o s a t i o n , which forms dimethylnitrosamine. There have been several epidemiological and several case r e ports inversely r e l a t i n g ascorbic acid intake from food to human cancer m o r t a l i t y . These studies are i n t e r e s t i n g , but may be confounded with the fact that the same ascorbic acid containing foods, namely f r u i t s and vegetables, also contain large amounts of vitamin A and f i b e r . Both vitamin A and f i b e r have been inversely related to human cancer mortality and have been shown to i n h i b i t several types of chemically-induced carcinogenesis in animals. Therefore, the possible anticancer effect of ascorbic acid may be due to other factors. 2

BHA and BHT Even though BHA (butylated hydroxyanisole) and BHT (butylated hydroxytoluene) are not naturally occurring antioxidants, various amounts of these compounds are added to food as food preservatives in order to reduce oxidative r a n c i d i t y . Both BHA and BHT are i n cluded in the FDA l i s t of substances generally accepted as safe (GRAS) and many acute and chronic tests have been done. Based on the evidence from these s t u d i e s , the FDA in 1977 recommended that BHT be removed from the GRAS l i s t and proposed interim s t u d i e s . BHA has been demonstrated to be an important i n h i b i t o r of carcinogenesis and has been extensively studied for i t s a b i l i t y to i n h i b i t carcinogen-induced neoplasia. Table I l i s t s several e x p e r i ments i n which BHA was administered before or during carcinogen exposure (27).

9.

SHAMBERGER

Publication Date: May 6, 1985 | doi: 10.1021/bk-1985-0277.ch009

Table I .

Antioxidants and Malonaldehyde in Cancer

Inhibition of Carcinogen-Induced Neoplasia by BHA

Carcinogen i n h i b i t e d

Species

S i t e of Neoplasm

Benzo(a)pyrene Benzo(a)pyrene Benzo(a)pyrene-7,8-dehydrodiol

Mouse Mouse Mouse

7,12-Dimethylbenz(a)anthracene 7,12-Dimethylbenz(a)anthracene 7,12-Dimethylbenz(a)anthracene 7,12-Di methylbenz(a)anth racene 7-Hydroxymethyl-12-methylbenz(a)anthracene Dibenz(a)anthracene Ni trosodi ethyl ami ne 4-Ni troqui noli ne-N-oxi de Uracil mustard Urethan Methylazoxymethanol acetate trans-5-Amino-3[2-(5-nitro-2furyl)vinyl]-l,2,4-oxadiazole

Mouse Mouse Mouse Rat

Lung Forestomach Forestomach, lung and lymphoid ti< Lung Forestomach Skin Breast

Mouse Mouse Mouse Mouse Mouse Mouse Mouse

Lung Lung Lung Lung Lung Lung Large i n t e s t i n e

Mouse

Forestomach, lung and lymphoid t i s s u e

It i s believed that BHA i n h i b i t s chemically induced carcinogenesis by producing a coordinated enzyme response that may be interpreted as causing a greater rate of d e t o x i f i c a t i o n (28). In a d d i t i o n , i n creased glutathione s-transferase and glutathione levels have been observed i n mice that have been fed BHA f o r 1-2 weeks i n carcinogen i n h i b i t i o n experiments (29). Glutathione s-transferase i s known t o be an important enzyme f o r detoxifying chemical carcinogens (2829). The anticarcinogenicity of BHA and BHT i n many experiments seems t o depend on the relationship of the time of administration of the carcinogen and BHA or BHT administration. If BHT was given before carcinogen administration, then i n h i b i t i o n of carcinogenesis occurs. However, i f BHT was given after the carcinogen, then enhancement of carcinogenesis occurs. Three groups of A/J mice were injected with urethan, 3-methylcholanthrene, or nitrosodimethylamine and then repeated doses of BHT. With a l l three carcinogens BHT treatment a f t e r carcinogen treatment s i g n i f i c a n t l y increased the numbers of lung tumors (30). Malonaldehyde Malonaldehyde, a three-carbon dialdehyde (0HC-CH -CH0), i s produced during l i p i d peroxidation by the oxidative decomposition of arachi donic and other unsaturated f a t t y a c i d s . Malonaldehyde i s present in a number of food products and i t s concentration i s increased by i r r a d i a t i o n of c e l l u l a r amino a c i d s , carbohydrates, deoxyribose, and DNA. Recent surveys (31-32) have confirmed the presence of malonaldehyde i n supermarket samples of meat, p o u l t r y , and f i s h , 2

115

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XENOBIOTIC METABOLISM: NUTRITIONAL EFFECTS

which constitute the main sources of malonaldehyde in the North American diet. Fruits and vegetables, in general, do not contain detectable amounts of malonaldehyde. There is also evidence that malonaldehyde is produced in vivo when there is an inadequate intake of vitamine E (33), which serves as a lipid antioxidant. Evidence has been reported on the formation of malonaldehyde in vivo during prostaglandin synthesis. Malonaldehyde is also formed on cellular exposure to ozone, carbon tetrachloride, ethanol and hydrocarbon compounds. Malonaldehyde (34-36) and the sodium form (37-38) have been shown to be mutagenic in the Salmonella test system, in L 5178 lymphoma cells (39), in rat skin fibroblasts (40), Drosophilia (41), and the Muller-5 sex-linked recessive lethaT mutation system"T41). Malonaldehyde also has some weak carcinogenic activity under some circumstances. Shamberger et. al. have found malonaldehyde to be an initiator in a malonaldehyde-croton oil test system (42). However, Fischer et. al. have found the sodium form of malonaldehyde to have neither initiating nor promoting activity (43). The sodium form of malonaldehyde also has been shown to increase the number of liver lesions in mice (44). Whether or not malonaldehyde is an important factor in the cancer process is not known. However, malonaldehyde is known to cross-link both protein and DNA. In general, unsaturated fat has more tumor-enhancing properties in many systems. However, there is no certain mechanism by which the breakdown of cell membrane unsaturated fatty acids might damage genetic material. In humans, about 50-60% of the ingested malonaldehyde from meat is excreted in the urine (45). It is not known how the remainder of the malonaldehyde is metabolized. The relative importance of the mutagenicity of malonaldehyde in human food is unknown. Certainly pyrolyzed food contains complete carcinogens such as benzopyrene and mutagenic substances such as tryptophan pyrolysates (Trp-P-1 and Trp-P-2), glutamic acid pyrolysates (Glu-P-1 and Glu-P-2), lysine pyrolysate (Lys-P-1), phenylalanine pyrolysate (Phe-P-1), and protein pyrolysates from broiled sardines (IQ and MelQ) and from broiled beef (MelQx). It is likely that antioxidants such as selenium and vitamins C and E also reduce the carcinogenic and mutagenic effect of these substances formed from pyrolyzed food in the same way that these antioxidants reduce the mutagenicity of malonaldehyde (46). Certainly more research is needed in this area. Literature Cited 1. 2. 3. 4. 5.

Shamberger, R.J., J. Nat. Cancer Inst. 1970, 44, 931-936. Riley, J.F., Experientia 1968, 15, 1237-1238. Clayton, C.C. and Baumann, C.A., Cancer Res. 1949, 9, 575-582. Griffin, A.C. and Jacobs, M.M., Cancer Lett. 1977, 3, 177-181. Dzhoiev, F.D., In Kantserog N-Nitrozosoedm: Deistvie, Obraz., Mater Simp., 3rd Tallinn, USSR, 1978; pp 51-53. 6. Harr, J.R., Exon, J.H., Weswig, P.H., and Whanger, P.P. Clin. Toxicol. 1973, 8, 487-495. 7. Marshall, M.V., Arnott, M.S., Jacobs, M.M., and Griffin, A.C., Cancer Lett. 1979, 7, 331-338. 8. O'Conner, T.P., Youngman, L.D., and Campbell, T.C., Fed. Proc. 1983, 42, 670.

Publication Date: May 6, 1985 | doi: 10.1021/bk-1985-0277.ch009

9. SHAMBERGER

Antioxidants and Malonaldehyde in Cancer

9. Baldwin, S., Parker, R.S., and Misslbeck., Fed. Proc. 1983, 42, 1312. 10. LeBoeuf, R.A., Laishes, B.A., and Hoekstra, W.G., Fed. Proc. 1983, 42, 669. 11. Schrauzer, G.N., and Ishmael, D., Ann. Clin. Lab. Sci. 1974, 4, 411-467. 12. Thompson, H.J., and Tagliaferro, A.R., Fed. Proc. 1980, 39, 1117. 13. Ip, C., Cancer Res. 1981, 41, 4386-4390. 14. Shamberger, R.J. and Willis, C.E. CRC Crit. Rev. Clin. Lab. Sci. 1971, 2, 211-221. 15. Shamberger, R.J., Tytko, S.A., and Willis, C.E., Arch. Environ. Health 1976, 31, 231-235. 16. Schrauzer, G.N., White, D.A., and Schneider, C.J., Bioinorg. Chem. 1977, 7, 23-24. 17. Ip, C., Carcinogenesis 1982, 3, 1453-1456. 18. Shklar, G.J., Natl. Cancer Inst. 1982, 68, 791-797. 19. Konings, A.W.T. and Trieling, W.B., Int. J. Radiat. Biol. 1977, 31, 397-400. 20. Shamberger, R.J., J. Natl. Cancer Inst. 1972, 48, 1491-1497. 21. Slaga, T.J. and Bracken, W.M., Cancer Res. 1977, 37, 1631-1635. 22. Sadek, I.A. and Abdelmegid, N., Oncology 1982, 39, 399-400. 23. Schlegel, J.U., Pipkin, G.E., Nishumura, R., and Schultz, G.N., Trans. Am. Assoc. Genitourinary Surg. 1969, 61, 85-89. 24. Mirvish, S.S., Wallace, L., Eagen, M. and Shubik, P., Science 1972, 177, 65-68. 25. Archer, M.C., Tannenbaum, S.R., Tan, T., and Weisman, M., J. Natl. Cancer Inst. 1975, 54, 1203-1205. 26. Chan, W.C. and Fong, Y.Y., Int. J. Cancer 1970, 20, 268-270. 27. Wattenberg, L.W. "In Environmental Carcinogenesis", Emmelot, P. and Kriek, E. Elsevier/North Holland Biomedical Press, Amsterdam, 1979, pp. 241-263. 28. Wattenberg, L.W., "In Cancer: Achievements, Challenges, and Prospects for the 1980's", Burchenol, J.H. and Oettgen Eds. Vol 1. Grune and Stratton, New York, 1981, pp. 517-539. 29. Benson, S.M., Cha, Y.N., Bueding, E., Heine, H.S., and Talalay, P., Cancer Res. 1979, 39, 2971-2977. 30. National Cancer Institute. Technical Report Series number 150. NIH Publ. No. 79-1706, Bethesda, Maryland: Carcinogenesis Testing Program, National Cancer Institute. 31. Shamberger, R.J., Shamberger, B.A., and Willis, C.E., J. Nutr. 1977, 107, 1404-1409. 32. Siu, G.M., and Draper, H.H., J. Food Sci. 1978, 43, 1147-1149. 33. Trostler, N., Brady, P.S., Romsos, D.R., and Leveille, G.A., J. Nutr. 1979, 109, 345-352. 34. Mukai, F.H. and Goldstein, B.D., Science, 1976, 191, 868-869. 35. Muchielli, A., 1975, Thesis, Univ. of Lille, CNRS, AO 11792. 36. Lawrence, M.J. and Tuttle, M.R., Cancer Res. 1980, 40, 276-282. 37. Marnett, L.J. and Tuttle, M.A., Cancer Res. 1980, 40, 276-282. 38. Basu, A.K. and Marnett, L.J., Carcinogenesis, 1983, 4, 331-334. 39. Yau, T.M., Mech Aging Dev., 1979, 11, 137-144. 40. Bird, R.P. and Draper, H.H., J. Toxicol. Environ. Hlth, 1980, 6, 811-823.

117

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XENOBIOTIC METABOLISM: NUTRITIONAL EFFECTS

41. Szabad, J., Soos, I., Polgar, G., Heijja, G., Mutation Research 1983, 113, 117-133. 42. Shamberger, R.J., Andreone, T.L., and Willis, C.E., J. Nat. Cancer Inst. 1974, 53, 1771-1773. 43. Fischer, S.M., Cancer Letters, 1983, 19, 61-66. 44. Bird, R.P., Draper, H.H., and Valli, V.E.O., J. Toxicol. and Environ. Hlth, 1982, 10, 897-905. 45. Jacobson, E.A., Newmark, H.L., Bird, R.P., and Bruce, W.R., Nutr. Rep. Int., 1983, 28, 509-517. 46. Shamberger, R.J., Corlett, C.L., Beaman, K.D. and Kasten, B.L., Mutat. Res. 1979, 66, 349-356.

Publication Date: May 6, 1985 | doi: 10.1021/bk-1985-0277.ch009

RECEIVED September 5, 1984

10 Influence of Types and Levels of Dietary Fat on Colon Cancer BANDARU S. REDDY

Publication Date: May 6, 1985 | doi: 10.1021/bk-1985-0277.ch010

Division of Nutrition and Endocrinology, Naylor Dana Institute for Disease Prevention, American Health Foundation, Valhalla, NY 10595 Epidemiologic studies indicate that diets high in total fat and saturated fat and low in certain fibers are associated with an increased risk for colon cancer. In addition, certain dietary fibers and cruciferous vegetables have been associated with a reduced risk in several populations consuming the diets high in total fat. In animal models, high dietary fat (corn o i l , safflower oil, beef fat and lard) increased the development of chemically-induced colon tumors; at high dietary fat levels, the types of fat (corn o i l , safflower o i l , lard and beef tallow) had no effect. However, diets high in saturated and monounsaturated fats of vegetable origin (coconut oil and olive oil) induced fewer colon tumors than the diets high in polyunsaturated fats (corn oil and saf flower oil). Thus, the fatty acid composition is one of the important factors in colon tumor promotion. The effect of dietary fat in colon cancer has been shown to be primarily during the post-initiation phase of carcinogenesis. Colon cancer is one of the most common tumors observed in the western population, exhibiting more than a tenfold excess when compared to the rural populations in Africa, Asia and South America. (1~3)« During the past several years, epidemiologic studies have revealed that our lifestyles, including dietary and nutritional practices, are important variables. These studies also suggested that not only the diets particularly high in total fat and low in certain dietary fibers, vegetables, and micronutrients are generally associated with an increased incidence of colon cancer in man, but dietary fat may be a risk factor in the absence of factors that are protective, such as use of high fibrous foods and fiber (4-11). However, the conduct and interpretation of epidemiologic studies is complicated by inherent problems in testing the dietary practices for their reliability, validity and sensitivity to reveal narrow but biologically significant differences, and to achieve some degree of dose stratification. When another line of evidence based on experimental studies, 0097-6156/85/0277-0119$06.00/0 © 1985 American Chemical Society

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EFFECTS

which have c o n s i s t e n t l y supported human e p i d e m i o l o g i c studies s u p p o r t s t h a t d i e t p l a y s an important r o l e i n the e t i o l o g y of c o l o n cancer, the r e l a t i o n s h i p between d i e t and c o l o n cancer deserves immediate a t t e n t i o n (_7).

Publication Date: May 6, 1985 | doi: 10.1021/bk-1985-0277.ch010

N u t r i t i o n a l Epidemiologic

Studies

Cancer of the c o l o n has been the s u b j e c t of s e v e r a l e p i d e m i o l o g i c reviews.(2,3,12). The v a r i a b i l i t y i n c o l o n c a n c e r i n c i d e n c e between c o u n t r i e s has d i r e c t e d r e s e a r c h toward s p e c i f i c environmental d i e t a r y f a c t o r s t h a t a r e c h a r a c t e r i s t i c of h i g h - r i s k p o p u l a t i o n . While some of t h e s e d i f f e r e n c e s may be due to g e n e t i c f a c t o r s o r l o c a l e n v i r o n m e n t a l f a c t o r s , f u r t h e r e v i d e n c e f o r the r o l e of d i e t ary environmental f a c t o r s i n c o l o n c a n c e r has been p r o v i d e d by m i g r a n t s t u d i e s which demonstrate a h i g h e r c o l o n cancer i n c i d e n c e r a t e i n the f i r s t and second g e n e r a t i o n Japanese immigrants to the U n i t e d S t a t e s and i n P o l i s h immigrants to A u s t r a l i a than i n Japanese (13). F u r t h e r m o r e , t i m e - t r e n d i n Japan showing t h a t c o l o n cancer seems to be i n c r e a s i n g i s c o n s i s t e n t w i t h the i n c r e a s i n g w e s t e r n i z a t i o n of the Japanese d i e t ( 1 4 ) . These s t u d i e s l e d s e v e r a l i n v e s t i g a t o r s to a c c e p t d i e t as a major e t i o l o g i c f a c t o r i n c o l o n c a n c e r . Wynder and Shigematsu (15) were the f i r s t to suggest t h a t n u t r i t i o n a l f a c t o r s i n g e n e r a l and s p e c i f i c a l l y d i f f e r e n c e s i n f a t i n take may be r e s p o n s i b l e f o r the i n t e r n a t i o n a l v a r i a t i o n i n c o l o n cancer incidence. Subsequent d e s c r i p t i v e e p i d e m i o l o g i c s t u d i e s have found a s t r o n g p o s i t i v e a s s o c i a t i o n between c o l o n c a n c e r m o r t a l i t y o r i n c i d e n c e i n d i f f e r e n t c o u n t r i e s and per c a p i t a a v a i l a b i l i t y i n n a t i o n a l d i e t s of t o t a l f a t (4,16) and of animal f a t , e s t i m a t e d from food balance sheets. Such international c o r r e l a t i o n s may be s u p p o r t i v e of a h y p o t h e s i s , but they s h o u l d be i n t e r p r e t e d w i t h c a u t i o n because the d i e t a r y d a t a were based not on a c t u a l i n t a k e i n f o r m a t i o n but on food d i s a p p e a r a n c e data. The n u t r i t i o n a l e p i d e m i o l o g i c s t u d i e s t u r n e d to c a s e - c o n t r o l comparisons and p r o s p e c t i v e s t u d i e s i n o r d e r to a c c u r a t e l y d e f i n e the e t i o l o g i c f a c t o r s . Wynder e t a l . (9^) conducted a l a r g e - s c a l e retrospective study on colon cancer patients i n Japan, which suggested a c o r r e l a t i o n between the w e s t e r n i z a t i o n of the Japanese d i e t and d i e t a r y f a t and c o l o n c a n c e r . A r e c e n t c a s e - c o n t r o l study i n A t h e n s , G r e e c e , demonstrated a p o s i t i v e a s s o c i a t i o n between c o l o n cancer and consumption of meat, but not o l i v e o i l ( 1 7 ) . In a n o t h e r study, no a s s o c i a t i o n was found w i t h i n c o u n t r i e s of r e g i o n a l or e t h n i c c o l o n cancer r a t e s i n r e l a t i o n to meat ( 1 8 ) . These c o n f l i c t ing results c o u l d be e x p l a i n e d on the b a s i s t h a t s e v e r a l of these s t u d i e s n e g l e c t e d to take i n t o c o n s i d e r a t i o n the o t h e r confounding f a c t o r s such as consumption of c r u c i f e r o u s v e g e t a b l e s , d i e t a r y f i b e r and o t h e r food items t h a t have been shown to reduce the r i s k of c o l o n cancer. F a i l u r e to f i n d c o n s i s t e n t s t r o n g r e l a t i o n s h i p s does not n e c e s s a r i l y mediate a g a i n s t a d i e t a r y e t i o l o g y of c o l o n c a n c e r , however, because c e r t a i n f i n d i n g s may have a r i s e n , a t l e a s t i n p a r t , from m e t h o d o l o g i c a l l i m i t a t i o n of these s t u d i e s . F i n a l l y , some of these s t u d i e s may have been hampered by the p o s s i b i l i t y t h a t d i e t s w i t h i n communities have been too u n i f o r m to permit a s s o c i a t i o n s between d i e t and d i s e a s e to be d e t e c t e d ( 1 9 ) .

Publication Date: May 6, 1985 | doi: 10.1021/bk-1985-0277.ch010

10.

REDDY

Influence of Dietary Fat on Colon Cancer

121

A c a s e - c o n t r o l study i n Canada i n d i c a t e d an e l e v a t e d r i s k f o r those w i t h an i n c r e a s e d i n t a k e of c a l o r i e s , t o t a l f a t , and s a t u r a t e d f a t (20,21). T h i s study e s t i m a t e d l e v e l s o f f a t consumption by comb i n i n g i n f o r m a t i o n from d i e t h i s t o r i e s w i t h i n f o r m a t i o n on the f a t content of foods. A r e c e n t c a s e - c o n t r o l study i n Utah Mormons i n d i c a t e d a p o s i t i v e a s s o c i a t i o n between d i e t a r y f a t and c o l o n c a n c e r (10). On the o t h e r hand, i n s e v e r a l p o p u l a t i o n s consuming the d i e t s h i g h i n t o t a l f a t , d i e t a r y f i b e r a c t s as a p r o t e c t i v e f a c t o r f o r c o l o n cancer r i s k . Recent s t u d i e s comparing r u r a l and urban p o p u l a t i o n s i n F i n l a n d , Denmark and Sweden and urban p o p u l a t i o n s i n New York i n d i c a t e d t h a t one of the f a c t o r s c o n t r i b u t i n g to the low r i s k of c o l o n cancer i n r u r a l S c a n d i n a v i a appears to be h i g h - d i e t a r y f i b e r i n t a k e , mainly whole-grain c e r e a l s , although a l l populations a r e on a h i g h - f a t d i e t ( 3 , 2 2 - 2 4 ) . A strong negative a s s o c i a t i o n was r e p o r t e d between r e g i o n a l c o l o n c a n c e r m o r t a l i t y w i t h i n the U n i t e d Kingdom and consumption of f i b e r foods c o n t a i n i n g h i g h amount of pentose ( 2 5 ) . The bulk of n u t r i t i o n a l e p i d e m i o l o g i c e v i d e n c e suggests t h a t d i e t s h i g h i n t o t a l f a t and low i n f i b e r are a s s o c i a t e d w i t h an i n c r e a s e d r i s k of c o l o n cancer i n man. In s e v e r a l p o p u l a t i o n s consumi n g a h i g h amount of t o t a l f a t , c e r t a i n d i e t a r y f i b e r s and c r u c i f e r ous v e g e t a b l e s a c t as p r o t e c t i v e f a c t o r s . Moreover, l a b o r a t o r y a n i mal s t u d i e s d i s c u s s e d elsewhere have c l e a r l y demonstrated t h a t h i g h f a t i n t a k e promotes the development of c o l o n c a n c e r . Concurrence between the nutritional epidemiologic and laboratory evidence o f f e r e d the s t r e n g t h to the concept t h a t d i e t i s a major e t i o l o g i c factor i n colon cancer. Mechanisms o f d i e t a r y f a t i n c o l o n

cancer

Food c o n t a i n s a l a r g e number of i n h i b i t o r s of c a r c i n o g e n e s i s , i n c l u d i n g f i b e r s , phenols, i n d o l e s , a r o m a t i c isothiocyanates, plant s t e r o l s , selenium s a l t s , a s c o r b i c a c i d , t o c o p h e r o l s and carotenes (26). These compounds have been shown to i n h i b i t neoplasms i n a n i mal models (26,27). Since the p r i n c i p a l s o u r c e s of these compounds i n the d i e t a r e p l a n t c o n s t i t u e n t s , the type and q u a n t i t y of the p l a n t m a t e r i a l i n the d i e t w i l l be of g r e a t importance i n d e t e r m i n i n g the a c t i v i t y of the p r o t e c t i v e system. Thus, the humans consumi n g r e l a t i v e l y l a r g e amounts of v e g e t a b l e s , b o t h c r u c i f e r o u s and n o n - c r u c i f e r o u s t y p e , and f r u i t s would have g r e a t e r defenses a g a i n s t c a r c i n o g e n s than do i n d i v i d u a l s consuming a l e s s e r amount of t h e s e foods. C u r r e n t l y , much of our knowledge on the mechanism of d i e t a r y f a t on c o l o n c a r c i n o g e n e s i s i s based on e x p e r i m e n t s conducted i n humans ( m e t a b o l i c e p i d e m i o l o g y ) and a n i m a l models ( 2 7 ) . The major s i g n i f i c a n c e of these s t u d i e s i s t h a t the primary e f f e c t of d i e t a r y f a t appears to be d u r i n g the p r o m o t i o n a l phase of c a r c i n o g e n e s i s r a t h e r than d u r i n g i n i t i a t i o n phase ( 2 8 ) . The amount of d i e t a r y f a t modulates the c o n c e n t r a t i o n of i n t e s t i n a l b i l e a c i d s as w e l l as the m e t a b o l i c a c t i v i t y of gut m i c r o f l o r a , w h i c h , i n t u r n , m e t a b o l i z e these s t e r o l s and o t h e r s u b s t a n c e s i n t o t u m o r i g e n i c compounds i n the c o l o n (29,30,31). These s t u d i e s have demonstrated t h a t h i g h f a t d i e t s i n c r e a s e the e x c r e t i o n of b i l e a c i d s i n t o the g u t . These b i l e

122

XENOBIOTIC M E T A B O L I S M : NUTRITIONAL

EFFECTS

a c i d s have been shown to a c t as c o l o n tumor promoters but do not have the p r o p e r t i e s of g e n o t o x i c c a r c i n o g e n s ( 3 1 ) . T h i s i s import a n t s i n c e c u r r e n t views on p r o p e r t i e s of promoters note t h a t the e f f e c t of such agents i s h i g h l y dependent on dose and on l e n g t h of e x p o s u r e , and thus p r o v i d e s an o p p o r t u n i t y of r e d u c i n g the r i s k of c o l o n c a n c e r development by l o w e r i n g the c o n c e n t r a t i o n of b i l e a c i d s by d i e t a r y means.

Publication Date: May 6, 1985 | doi: 10.1021/bk-1985-0277.ch010

Metabolic

(Biochemical) Epidemiologic

S t u d i e s on D i e t a r y F a t

The concept t h a t d i e t a r y f a t and c e r t a i n f i b e r s d i s t i n c t from chemic a l c o n t a m i n a n t s of d i e t and from o t h e r e n v i r o n m e n t a l and g e n e t i c f a c t o r s a r e important determinants of c o l o n c a n c e r r i s k i s r e i n f o r c e d by b i o c h e m i c a l e p i d e m i o l o g i c s t u d i e s i n humans and l a b o r a t o r y animal s t u d i e s . A key i n s i g h t gained from s t u d i e s i n man i s t h a t the c o n c e n t r a t i o n of t o t a l b i l e a c i d s and i n d i v i d u a l b i l e a c i d s , namely d e o x y c h o l i c acid and lithocholic a c i d , i s much lower i n s t o o l s from l o w - r i s k p o p u l a t i o n s such as Japanese and o t h e r A s i a n s and A f r i c a n s consuming a l o w - f a t d i e t when compared to h i g h - r i s k p o p u l a t i o n s such as North Americans and w e s t e r n Europeans consuming a h i g h - f a t d i e t (29,31,32). People on a h i g h - f a t d i e t appear to have h i g h e r l e v e l s of f e c a l secondary b i l e a c i d s compared to t h o s e on a l o w - f a t d i e t . In g e n e r a l , t h e r e a r e no major d i f f e r e n c e s i n the f e c a l m i c r o f l o r a p r o f i l e s of these d i f f e r e n t r i s k groups a l though the m e t a b o l i c a c t i v i t y of some of the c o n s t i t u e n t m i c r o f l o r a , p a r t i c u l a r l y the n u c l e a r d e h y d r o g e n a t i n g C l o s t r i d i a and bacterial enzymes such as p - g l u c u r o n i d a s e and 73CH

Figure

to tumorigenesis.

Propyl Gallate. compounds u s e d i n t h e s e

Publication Date: May 6, 1985 | doi: 10.1021/bk-1985-0277.ch011

11.

KING ET A L .

Modulation of Mammary Tumor Incidence

135

s t e e l e a r t a g w i t h a d i s t i n c t i v e and s e q u e n t i a l number f o r i d e n t i f i cation. A l l animals were housed i n an AAALAC-accredited animal c a r e f a c i l i t y w i t h a c o n t r o l l e d temperature (68-72°) and h u m i d i t y (50-60%) and a 12 h r l i g h t / d a r k c y c l e . A l l e x p e r i m e n t a l animals were weighed weekly from the time o f a r r i v a l and p a l p a t e d weekly f o r tumors b e g i n n i n g t h r e e weeks a f t e r c a r c i n o g e n t r e a t m e n t . Only tumors v e r i f i e d h i s t o l o g i c a l l y as mammary adenocarcinomas were i n c l u d e d i n incidence data. Animals used f o r s t u d y i n g drug m e t a b o l i s m and measuring v a r i o u s microsomal parameters were p l a c e d on d i e t s , weighed and t r e a t e d i n e x a c t l y the same manner as those b e i n g used f o r mammary tumor i n c i d e n c e s t u d i e s , except t h a t they were k i l l e d a t 50 days o f age, the time f o r exposure o f r a t s i n the o t h e r e x p e r i m e n t a l group t o the carcinogen. L i v e r s and/or mammary g l a n d s were h a r v e s t e d f o r p r e p a r a t i o n o f s u b c e l l u l a r f r a c t i o n s and drug m e t a b o l i s m s t u d i e s as p r e v i ously d e s c r i b e d (28). A n t i o x i d a n t measurements i n t i s s u e s were made u s i n g s i m i l a r animals t h a t had been f e d the b a s i c d i e t s supplemented w i t h a n t i o x i d a n t s f o r the s p e c i f i c times as i n d i c a t e d f o r each experiment. R e s u l t s and D i s c u s s i o n Over the p a s t s e v e r a l y e a r s the DMBA-induced r a t mammary tumor model has been t e s t e d many times w i t h v a r i o u s s p e c i f i c changes i n the e x p e r i m e n t a l parameters b e i n g t e s t e d . The v e r y f i r s t i n c i d e n c e s t u d y u t i l i z e d the same t h r e e b a s i c d i e t s ( i n c o r p o r a t i n g the H u b b e l l s a l t mix) w i t h and without 0.7% BHT s u p p l e m e n t a t i o n ( 1 3 ) . I t was found i n t h i s study t h a t BHT g i v e n a t t h i s l e v e l , a l t h o u g h an e f f e c t i v e tumor i n h i b i t o r , was a l s o s l i g h t l y t o x i c , r e s u l t i n g i n a s l i g h t d e c r e a s e i n weight g a i n and o c c a s i o n a l and i n t e r m i t t e n t h a i r l o s s . T h i s study was r e p e a t e d u s i n g the same d i e t s , b u t w i t h o n l y 0.3% BHT supplementation. Almost i d e n t i c a l tumor i n c i d e n c e and tumor numbers were o b t a i n e d u s i n g t h i s l e v e l o f BHT, but weight g a i n s were now the same as i n non-BHT-supplemented animals and no e v i d e n c e was seen o f any toxic side e f f e c t s . The same p r o t o c o l was employed f o r t e s t i n g each o f the f o l l o w i n g : BHA, a - t o c o p h e r o l (as a - t o c o p h e r y l a c e t a t e ) and p r o p y l g a l l a t e (PrG) as mammary tumor i n h i b i t o r s . Weight g a i n s i n a l l groups ± a n t i o x i d a n t s were e s s e n t i a l l y i d e n t i c a l and no t o x i c s i d e e f f e c t s were o b s e r v e d . F i g u r e 3 demonstrates the tumor i n c i d e n c e r e s u l t s o b t a i n e d i n these s t u d i e s when j u s t the t h r e e d i e t a r y regimens were compared, i . e . LF ( 2 % ) , HPF (20% s t r i p p e d c o r n o i l ) and HSF (18% s t r i p p e d c o r n o i l ± 2% l i n o l e i c a c i d ) . As c a n be seen i n t h i s f i g u r e , the animals consuming the HPF d i e t r o u t i n e l y d e v e l o p a 97-100% tumor i n c i d e n c e . Those on the HSF d i e t d e v e l o p a 55-70% i n c i d e n c e and those on the L F d e v e l o p o n l y a 20-30% i n c i d e n c e o f mammary tumors. These v a l u e s r e p r e s e n t t h r e e s e t s o f experiments w i t h 30 r a t s p e r group o r 90 t o t a l r a t s per each d i e t r e p r e s e n t e d . There i s a 10-15% v a r i a b i l i t y i n the i n c i d e n c e r a t e s between experiments performed under i d e n t i c a l c o n d i t i o n s whether done c o n c u r r e n t l y o r i n a sequence o f t i m e s . These r e s u l t s have been used as the s t a n d a r d f o r comparison o f a d d i t i o n a l experiments, and those b e i n g performed most r e c e n t l y compare w e l l with the e a r l i e r experiments. Thus, t h i s seems t o be a s a t i s f a c t o r i l y r e p r o d u c i b l e model.

Publication Date: May 6, 1985 | doi: 10.1021/bk-1985-0277.ch011

136

XENOBIOTIC M E T A B O L I S M : N U T R I T I O N A L E F F E C T S

F o r v a r i o u s reasons over the past t e n y e a r s , p r i m a r i l y i n attempt to improve the b i o l o g i c a l d e s i g n , changes have been made i n the o r i g i n a l b a s i c d i e t s . One o f the f i r s t changes was from the use of H u b b e l l S a l t Mix to the AIN 76 S a l t Mix. The changes caused no d e t e c t a b l e d i f f e r e n c e i n the r e s u l t s w i t h r e g a r d to tumor i n c i d e n c e or tumor numbers per t u m o r - b e a r i n g r a t . In some e x p e r i m e n t s , d i e t s have been i s o c a l o r i c a l l y p a i r - f e d to the LF d i e t , and more r e c e n t l y , a d j u s t e d so t h a t a l l d i e t s have e q u i v a l e n t n u t r i e n t - t o - c a l o r i e ratios. The l a s t adjustment was made ( T a b l e I I ) to compensate f o r the f a c t t h a t the animals which are f e d the LF d i e t s consume more d i e t ( i n g) to o b t a i n the same number c a l o r i e s a v a i l a b e to the HSF and HPF-fed a n i m a l s i n a much s m a l l e r amount o f d i e t . Through t h i s adjustment, a l l animals i n the v a r i o u s d i e t a r y groups w i l l r e c e i v e e s s e n t i a l l y the same amount o f t r a c e elements and v i t a m i n s . Our more r e c e n t s t u d i e s have been c a r r i e d out u s i n g these a d j u s t e d d i e t s ( T a b l e I I ) . When the BHT and BHA s t u d i e s were r e p e a t e d w i t h the v a r i o u s d i e t s , s i m i l a r r e s u l t s were o b t a i n e d compared to the e a r l i e r d i e t s ( T a b l e I and r e f e r e n c e 13). These r e s u l t s , a l o n g w i t h the companion r e s u l t s t h a t s i m i l a r tumor number and i n c i d e n c e were o b t a i n e d , support the h y p o t h e s i s t h a t the i n f l u e n c e o f d i e t on t h i s tumor model i s an e f f e c t o f the type and q u a n t i t y o f f a t f e d , and does not appear t o be a c a l o r i c e f f e c t . T h i s p o i n t has been r e p e a t e d l y s t r e s s e d i n both formal and i n f o r m a l s c i e n t i f i c exchanges o f v i e w s , and our work as w e l l as the work o f o t h e r s c o n t i n u e s to show t h a t c a l o r i c d e n s i t y per se i s not r e s p o n s i b l e f o r the enhancement o f t u m o r i g e n e s i s seen i n t h i s model.

Table I I . E x p e r i m e n t a l D i e t s (Equivalent N u t r i e n t : C a l o r i e Ratios) High Polyunsaturated Fat D i e t % by Casein Fat Sucrose Salt Mixture Alphacel (NonN u t r i e n t Bulk) Vitamin Mixture d

e

a

b

c

d

e

wt.

28.20 20.00 38.77 4.90 6.93 1.20

a

High Saturated Fat Diet % by

wt.

28.20 20.00 38.77 4.90

b

6.93 1.20

Low Fat Diet % by

wt.

23.0 2.0 64.0 4.0

C

6.0 1.0

S t r i p p e d corn o i l . 1 8 % coconut o i l + 2% l i n o l e i c a c i d methyl e s t e r s . 2 % l i n o l e i c a c i d methyl e s t e r s . AIN-76 m i n e r a l m i x t u r e . AIN-76 v i t a m i n m i x t u r e .

With the a d d i t i o n o f the d i f f e r e n t a n t i o x i d a n t s to the b a s i c s e m i p u r i f i e d d i e t s d i f f e r i n g i n f a t c o n t e n t , v a r i e d r e s u l t s have been obtained. Both BHT and PrG have r o u t i n e l y g i v e n good p r o t e c t i o n when

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K I N G ET A L .

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137

added t o the d i e t a t a l e v e l o f 0.3% (w/w). BHA and a - t o c o p h e r o l have shown o n l y m a r g i n a l o r no s i g n i f i c a n t p r o t e c t i o n a g a i n s t mammary adenocarcinomas ( F i g u r e 4 ) . The f a c t t h a t BHA showed no s i g n i f i c a n t p r o t e c t i o n a g a i n s t mammary t u m o r i g e n e s i s i n t h i s model was o f p a r t i c u l a r i n t e r e s t because o f i t s s i m i l a r i t y t o BHT, and because i t had been r e p o r t e d t o be an e f f e c t i v e tumor i n h i b i t o r i n o t h e r models o f c a r c i n o g e n e s i s (19) . Q u i t e a v a r i e t y o f a n t i o x i d a n t s have been t e s t e d and found t o be e f f e c t i v e i n s e v e r a l d i f f e r e n t models and t h e e f f e c t i s f a r from c o n s i s t e n t . A l l a n t i o x i d a n t s do not i n h i b i t any one tumor model and some tumor models do n o t appear t o be i n h i b i t e d by any a n t i o x i d a n t (20-22). I m p l i c i t i n many o f these s t u d i e s has been t h e assumption t h a t i t i s t h e a n t i o x i d a n t p r o p e r t i e s o f t h i s c l a s s o f p h e n o l i c compounds t h a t i s r e s p o n s i b l e f o r t h e i r tumor inhibitory capabilities. Certainly i n s u f f i c i e n t information i s a v a i l a b l e p r e s e n t l y t o make a d e c i s i o n one way o r a n o t h e r i n t h i s regard. This research area r e q u i r e s f u r t h e r i n v e s t i g a t i o n i n order to g a i n i n s i g h t s which may suggest the mechanisms i n v o l v e d i n r e g u l a t i n g tumor growth r a t h e r than approach t h e problem t o f i n d a c t u a l and p r a c t i c a l means o f c a n c e r p r e v e n t i o n . More r e c e n t l y , we have focused on t h e s t r a t e g y o f i n v e s t i g a t i n g the mechanism o f i n h i b i t i o n o f c a r c i n o g e n e s i s by BHT i n terms o f t h e a l t e r a t i o n s i n s p e c i f i c organ b i o c h e m i s t r y t h a t r e s u l t from f e e d i n g t h i s and o t h e r a n t i o x i d a n t s . In t h i s v e i n , we have been f o c u s i n g on the f o l l o w i n g experiments d e s i g n e d t o p r o v i d e i n f o r m a t i o n as t o t h e mechanisms o f tumor i n c i d e n c e / g r o w t h m o d i f i c a t i o n by d i e t a r y f a t and antioxidants: 1) i n v e s t i g a t i o n s t o determine how t h e l e v e l o f u n s a t u r a t i o n p e r se i n f l u e n c e s t h e e f f e c t i v e n e s s o f a n t i o x i d a n t s as i n h i b i t o r s o f mammary g l a n d t u m o r i g e n e s i s ; 2) i n v e s t i g a t i o n s t o d e t e r m i n e the e f f e c t o f time d u r i n g which t h e a n t i o x i d a n t t o be t e s t e d was s u p p l i e d t o t h e animal w i t h r e s p e c t t o t h e time o f exposure to the c a r c i n o g e n , DMBA; and 3) d e t e r m i n a t i o n s o f the a c t u a l t i s s u e c o n c e n t r a t i o n s o f the v a r i o u s a n t i o x i d a n t s with respect t o time o f f e e d i n g , and amount and type d i e t a r y f a t b e i n g f e d . The degree o f u n s a t u r a t i o n o f animal t i s s u e s i s a f u n c t i o n o f the l e v e l o f u n s a t u r a t e d f a t consumed i n the d i e t . Hence, a b a l a n c e between d i e t a r y f a c t o r s which e i t h e r f a c i l i t a t e s o r i n h i b i t s c a r c i n o gen metabolism may have an important e f f e c t on the e v e n t u a l outcome o f c h r o n i c c a r c i n o g e n exposure. One p o s s i b l e e f f e c t o f d i e t a r y l i p i d s and a n t i o x i d a n t s on c h e m i c a l c a r c i n o g e n e s i s t h a t must be cons i d e r e d i s t h e i n f l u e n c e o f these d i e t a r y components on t h e metabol i s m o f chemical carcinogens. A f t e r PAH's e n t e r t h e body, they a r e o x i d i z e d i n t h e endoplasmic r e t i c u l u m o f many t i s s u e s ( l i v e r , s k i n , mammary, e t c . ) t o h y d r o x y l a t e d p r o d u c t s which, b e i n g w a t e r - s o l u b l e , are more r e a d i l y e x c r e t e d ( F i g u r e 5 ) . U n f o r t u n a t e l y , i n t h i s p r o c e s s which u s u a l l y r e s u l t s i n d e t o x i f i c a t i o n , some compounds a r e c o n v e r t e d to r e a c t i v e a l k y l a t i n g agents which a r e c a r c i n o g e n i c . Benson o b s e r v e d t h a t mammary t i s s u e from animals on a h i g h f a t d i e t appeared more m e t a b o l i c a l l y a c t i v e when compared t o t i s s u e from animals b e i n g fed a low f a t d i e t ( 2 8 ) . More r e c e n t l y , Dao's l a b o r a t o r y has demons t r a t e d t h a t mammary g l a n d t i s s u e p e r se i s c a p a b l e o f m e t a b o l i z i n g DMBA, and that t h i s m e t a b o l i s m c a n be a f f e c t e d by known i n d u c e r s o f the h e p a t i c monooxygenase system such as 3 - m e t h y l c h o l a n t h r e n e ( 3 0 , 31). V i r g i n mammary glands from female Sprague-Dawley r a t s have been shown t o c o n t a i n an i n d u c i b l e cytochrome P-450 i n t h e m i c r o s o m a l monooxygenase system ( 2 9 ) . I t i s r e a s o n a b l e t o assume t h a t d i e t a r y

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XENOBIOTIC M E T A B O L I S M : N U T R I T I O N A L E F F E C T S

AGE

(weeks)

F i g u r e 3. I n c i d e n c e o f mammary tumors i n female Sprague-Dawley r a t s f e d d i e t s c o n t a i n i n g d i f f e r e n t amounts and types o f f a t . [•] 20% c o r n o i l f a t ; [A] 20% coconut o i l ; [•] 2% m e t h y l linoleate. See E x p e r i m e n t a l D e s i g n f o r d e t a i l s o f these d i e t s .

F i g u r e 4. D i e t a r y f a t and a n t i o x i d a n t mammary c a r c i n o g e n e s i s .

i n f l u e n c e s on M B A - i n d u c e d

11.

KING ET A L .

Modulation of Mammary Tumor Incidence CARCINOGEN

*

139

DETOXIFICATION

(PARENT COMPOUND)

ACTIVATION (AAH?)

? [~PROXIMATE CARCINOGEN] [_

(INTERMEDIATE)

(EH)

^ DETOXIFICATION

_ J OR (GSH)

CELLULAR TARGET(S) FURTHER METABOLISM

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ULTIMATE CARCINOGEN

•DETOXIFICATION OR (GSH)

Rx WITH CELLULAR TARGET(S)

Figure

5.

Scheme o f c a r c i n o g e n m e t a b o l i s m i n mammalian t a r g e

tissues.

f a t c a n a f f e c t c a r c i n o g e n metabolism i n the mammary g l a n d , s i n c e i t has been shown t o a f f e c t m i c r o s o m a l m e t a b o l i c a c t i v i t i e s i n o t h e r extra-hepatic tissues. F o r example, AHH a c t i v i t y has been shown t o i n c r e a s e i n the r a t k i d n e y as d i e t a r y f a t i s i n c r e a s e d above 4% i n the d i e t , r e a c h i n g a maximum a f t e r the d i e t s have been f e d f o r seven days ( 2 3 ) . A n o t h e r parameter o f the m e t a b o l i c p r o c e s s which c o u l d p o t e n t i a l l y be a f f e c t e d by d i e t a r y f a t s and a n t i o x i d a n t s i s a major component o f the endoplasmic r e t i c u l a r monooxygenase system, c y t o c h r o m e ( s ) P-450. These cytochromes a l o n g w i t h t h e i r a s s o c i a t e d enzyme NADPHcytochrome P-450 r e d u c t a s e , a r e a l s o l i k e l y t o be i n v o l v e d i n the oxidation of p o t e n t i a l l y carcinogenic substrates. This reductase i s a f l a v o p r o t e i n which t r a n s f e r s e l e c t r o n s from NADPH t o cytochrome P450 i n two s t e p s . The s e l e c t i v e i n d u c t i o n o f s e v e r a l l i v e r monooxygenases by BHT i s w e l l - e s t a b l i s h e d (25_, 3 4 ) . D i e t a r y BHT i n male r a t s f e d a chow d i e t a p p a r e n t l y produces a change i n t h e r e l a t i v e p r o p o r t i o n o f i n d i v i d u a l forms o f cytochrome P-450 w i t h o u t a l t e r i n g the t o t a l c o n c e n t r a t i o n o f cytochromes ( 2 6 ) . S i m i l a r l y , i n male r a t s fed e i t h e r the HPF o r HSF d i e t s , the s p e c i f i c c o n t e n t o f cytochrome P-450 i s s i g n i f i c a n t l y h i g h e r i n l i v e r microsomes from r a t s f e d t h e s e d i e t s compared t o the l o w - f a t f e d animals ( 2 9 ) . Whether the f a t was s a t u r a t e d o r p o l y u n s a t u r a t e d d i d not a f f e c t t h e s e m i c r o s o m a l components. With female r a t s , however, BHT produces an i n d u c t i o n o f l i v e r microsomal cytochrome P-450 c o n t e n t ( T a b l e I I I ) i n a n i m a l s consuming semipurified diets high i n either polyunsaturated or saturated f a t (27). Marked d e c r e a s e s i n NADPH cytochrome P-450 r e d u c t a s e a c t i v i t y were c o n s i s t e n t l y seen when BHT was i n c l u d e d i n t h e d i e t o f male r a t s (24) but no s i g n i f i c a n t e f f e c t s were noted i n female r a t s ( 2 7 ) .

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140

Reductase a c t i v i t i e s were o n l y 30-35% o f those i n microsomes from unsupplemented male a n i m a l s , the most profound d e c r e a s e o c c u r r i n g i n the l o w - f a t - f e d a n i m a l s . Reductase i s g e n e r a l l y c o n s i d e r e d t o be t h e r a t e - l i m i t i n g component o f the microsomal monooxygenase system and t h i s d e c r e a s e i n a c t i v i t y c o u l d have profound e f f e c t s on the metabol i s m o f these f o r e i g n compounds. Mammary AHH has been shown i n v i r g i n r a t s and found to v a r y as a f u n c t i o n o f the age o f the a n i m a l , b e i n g h i g h e s t i n younger a n i m a l s (30-60 days o l d ) (32, 3 3 ) . I n d u c t i o n o f t h i s enzyme i s a l s o agedependent, b e i n g h i g h l y i n d u c i b l e between 40 and 60 days o f age. AHH a c t i v i t y i s important from the s t a n d p o i n t t h a t t h i s type o f a c t i v i t y r e s u l t s i n the f o r m a t i o n o f h i g h l y r e a c t i v e e l e c t r o p h o l i c i n t e r m e d i a t e s , arene o x i d e s , from r e l a t i v e l y i n e r t p r e c u r s o r s . P r e l i m i n a r y d a t a from o u r l a b o r a t o r y s u g g e s t s t h a t AHH a c t i v i t y i s s p e c i f i c a l l y i n f l u e n c e d by both the l e v e l and type o f d i e t a r y f a t . As can be seen from T a b l e IV, h e p a t i c AHH a c t i v i t y measured as b e n z ( a ) p y r e n e h y d r o x y l a s e and read a g a i n s t s t a n d a r d 3-0H-benzpyrene, i s h i g h e s t i n the HPF d i e t a r y group f o l l o w e d by the HSF and lowest i n the L F - f e d group, and the lowest v a l u e f o r LF i s s i g n i f i c a n t l y induced by HPF (P < 0.05). A d d i t i o n o f BHT t o the d i e t a t a l e v e l o f 0.3% as b e f o r e , r e s u l t e d i n a p p r o x i m a t e l y a f o u r - t o f i v e f o l d i n c r e a s e i n AHH a c t i v i t y (Table IV). Thus, d i e t a l s o i n f l u e n c e s t h e AHH l e v e l w i t h the HPF v a l u e s i g n i f i c a n t l y (P < 0.05) induced above LF ( s t u d e n t ' s t test). T a b l e I I I . E f f e c t s o f D i e t a r y F a t and A n t i o x i d a n t s on H e p a t i c Microsomal Cytochrome P. L e v e l s i n Female Sprague-Dawley Rats 5

Diet

Q

T o t a l Cytochrome P ^ Q (n moles/mg MS P r o t e i n )

High P o l y u n s a t u r a t e d Fat •BHT •BHA •a-Toc •PrG

0.79 1.18 0.87 0.79 0.80

± + ± + +

0.04t 0.05* 0.06 0.05 0.04

High S a t u r a t e d F a t •BHT •BHA •a-Toc •PrG

0.85 1.10 0.73 0.74 0.77

+ + ± ± +

0.06 0.08* 0.07 0.06 0.03

Low F a t •BHT •BHA •a-Toc •PrG

0.81 0.92 0.76 0.78 0.74

+ ± ± ± ±

0.04 0.07 0.06 0.06 0.03

tMean ± SEM. * D i f f e r s i g n i f i c a n t l y from t h a t f o r the group on the c o r r e s p o n d i n g non-supplemented d i e t (P < 0.05 by s t u d e n t ' s t t e s t ) . BHT = b u t y l a t e d h y d r o x y t o l u e n e (0.3% by weight) BHA = b u t y l a t e d h y d r o x y a n i s o l e (0.3%) a-Toc = a - t o c o p h e r y l a c e t a t e (0.2%) PrG = p r o p y l g a l l a t e (0.3%)

11.

KING ET A L .

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141

T a b l e IV. A r y l Hydrocarbon Hydroxylase A c t i v i t y i n H e p a t i c Microsomes from Animals Consuming V a r i o u s L e v e l s and Types o f F a t ± 0 . 3 % BHT Polyunsaturated Fat -BHT +BHT

6.3 ± 0.4*t 26.5 ± 1.5

Saturated Fat 4.0 21.6

± 0.2 ± 1.0

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tMean ± S.E. o f 10-12 d e t e r m i n a t i o n s . *AHH a c t i v i t y measured as AOD/min/mg microsomal

Low 3.4 17.3

Fat ± 0.1 ± 0.9

protein.

Thus, i t c a n be e a s i l y t h e o r i z e d t h a t t h e s h i f t seen i n t h e microsomal enzyme and cytochrome p a t t e r n s caused by d i e t a r y f a t and a n t i o x i d a n t s may be r e l a t e d t o the i n h i b i t o r y and enhancing e f f e c t s o f these compounds on c h e m i c a l c a r c i n o g e n e s i s . F u r t h e r s t u d i e s a r e i n d i c a t e d t o l o o k a t s p e c i f i c c a r c i n o g e n m e t a b o l i t e s produced by t h e drug m e t a b o l i z i n g systems o f animals consuming the v a r i o u s d i e t s , and to attempt t o c o r r e l a t e t h e c o r r e s p o n d i n g cytochrome and enzyme l e v e l s w i t h these m e t a b o l i t e s and u l t i m a t e l y w i t h r e s u l t a n t tumor i n c i d e n c e s and tumor numbers. C r i t i c a l Time o f BHT E x p o s u r e . Two complimentary s t u d i e s were p e r formed w i t h t h e s p e c i f i c o b j e c t i v e o f d e t e r m i n i n g t h e c r i t i c a l time d u r i n g which BHT must be f e d t o r e a l i z e p r o t e c t i o n a g a i n s t DMBAi n d u c e d mammary c a r c i n o g e n e s i s . These s t u d i e s have been r e p o r t e d elsewhere (35) and w i l l o n l y be b r i e f l y summarized h e r e . The r e s u l t s were comparable i n both t h e HPF and HSF-supplemented groups. In t h e f i r s t study 0.3% BHT was added t o the HPF and HSF d i e t s a t v a r i o u s times from weaning (21 days o f age) t o one week b e f o r e c a r c i n o g e n a d m i n i s t r a t i o n (42 days o f a g e ) , a t 50 days o f age, t h e time o f DMBA t r e a t m e n t , o r one, two o r t h r e e weeks a f t e r DMBA, and i n c l u d e d i n t h e d i e t from t h a t time throughout the e x p e r i m e n t a l p e r i o d o f 30 weeks. In a s i m i l a r experiment, BHT was added t o t h e d i e t f o r o n l y s h o r t p e r i o d s o f time, one week s t a r t i n g a t weaning (21 days o l d ) then removed from t h e d i e t , o r added f o r two, t h r e e , f o u r , f i v e o r s i x weeks from weaning and then removed. T h i s encompasses the time from weaning up t o and i n c l u d i n g t h r e e weeks p o s t - c a r c i n o g e n treatment a f t e r each o f the r e s p e c t i v e BHT f e e d i n g p e r i o d s , the a n t i o x i d a n t was removed from t h e d i e t s and the animals c o n t i n u e d f o r t h e remainder o f the e x p e r i m e n t a l p e r i o d on t h e i r r e s p e c t i v e HPF o r HSF d i e t s . When mammary tumor i n c i d e n c e r a t e s were compared w i t h non-BHT t r e a t e d animals i t was found from the f i r s t experiment t h a t BHT was most e f f e c t i v e i f added t o the d i e t a t t h e time o f weaning o r up t o t h e time o f c a r c i n o g e n t r e a t m e n t . I f one w a i t e d u n t i l t h r e e weeks p o s t c a r c i n o g e n t o supplement t h e d i e t s w i t h BHT, e s s e n t i a l l y no p r o t e c t i o n was seen, i . e . the r e s u l t s were comparable t o those when no BHT was added t o t h e d i e t throughout the e x p e r i m e n t a l f e e d i n g time. In the s h o r t - t e r m BHT s u p p l e m e n t a t i o n experiment, i t was found t h a t no p r o t e c t i o n was o b t a i n e d i f BHT was added f o r o n l y 1 o r 2 weeks and then removed from the d i e t (2 weeks p r e - c a r c i n o g e n t r e a t ment). I f i t was c o n t i n u e d f o r t h r e e weeks ( t o 1 week p r e - c a r c i n o gen) , o n l y minimal p r o t e c t i o n was seen. I f f e d up t o the time o f DMBA-treatment o r f o r one, two o r t h r e e weeks p o s t - c a r c i n o g e n then removed, maximal p r o t e c t i o n was seen, comparable t o the group t h a t consumed BHT throughout t h e e n t i r e e x p e r i m e n t a l p e r i o d . These two

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EFFECTS

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s t u d i e s i n d i c a t e t h a t BHT needs t o be p r e s e n t i n t h e d i e t f o r twot h r e e weeks b e f o r e and a f t e r the time o f c a r c i n o g e n exposure t o be effective. S t o p p i n g i t too soon o r s t a r t i n g i t too l a t e a f t e r DMBA exposure does not a l l o w i t t o be e f f e c t i v e as a tumor i n h i b i t o r . These r e s u l t s i m p l i c a t e BHT as h a v i n g a r o l e i n the a c t i v a t i o n , i n i t i a t i o n o r e a r l y p r o m o t i o n a l phase o f DMBA-induced c h e m i c a l carcinogenesis. A d e f i n i t e p o s s i b i l i t y i s t h a t i t may r e q u i r e t h i s p e r i o d o f time t o a l l o w f o r the i n d u c t i o n o f s p e c i f i c m e t a b o l i c enzymes i n v o l v e d i n the h a n d l i n g o f DMBA. T h i s time may be r e q u i r e d f o r the a t t a i n m e n t o f s u f f i c i e n t l e v e l s i n the t i s s u e s t o a l l o w e f f e c t i v e n e s s as an i n h i b i t o r . F u r t h e r s t u d i e s a r e c e r t a i n l y needed to narrow the wide range o f p o s s i b i l i t i e s . Many q u e s t i o n s i n the i n v e s t i g a t i o n o f a p o s s i b l e mechanism f o r a n t i o x i d a n t i n h i b i t i o n o f mammary c a r c i n o g e n e s i s have l e d us t o t h e need t o be a b l e t o measure a c t u a l t i s s u e c o n c e n t r a t i o n s o f these compounds. Since BHT was found t o be a v e r y e f f e c t i v e mammary tumor i n h i b i t o r i n t h i s model, and BHA t o have no measurable e f f e c t under the same e x p e r i m e n t a l c o n d i t i o n s , one has t o a s k t h e q u e s t i o n as t o whether o r not both a r e absorbed and taken up by the t i s s u e s , s p e c i f i c a l l y mammary t i s s u e , s i n c e t h i s i s the t a r g e t t i s s u e . BHT was not e f f e c t i v e as an i n h i b i t o r when f e d f o r t h r e e weeks from weaning, then stopped one week p r i o r t o c a r c i n o g e n treatment ( 3 5 ) . BHT was e f f e c t i v e i n r e d u c i n g tumor development when added t o the d i e t two-three weeks b e f o r e and up t o the time o f c a r c i n o g e n expos u r e , and m a i n t a i n e d i n the d i e t from t h a t time on through the 30 week e x p e r i m e n t a l time. The e f f e c t i v e n e s s o f BHT i n i n h i b i t i n g t u m o r i g e n e s i s d e c r e a s e d as the time o f i n i t i a t i n g BHT f e e d i n g a f t e r c a r c i n o g e n treatment i n c r e a s e d , w i t h s i m i l a r r e s u l t s b e i n g o b t a i n e d i n both HPF and HSF-fed a n i m a l s . I t was o f i n t e r e s t t o study how BHT p r o t e c t s a g a i n s t mammary tumors, w h i l e BHA does n o t (13, 36). A l l t h e s e o b s e r v a t i o n s l e a d one t o ask "what a r e the a c t u a l t i s s u e c o n c e n t r a t i o n s o f BHT and BHA a f t e r b e i n g consumed at 0.3% o f the d i e t by weight f o r v a r i o u s p e r i o d s o f time? Are t h e y taken up e q u a l l y w e l l , o r at a l l by the mammary t i s s u e and/or the l i v e r ? 11

I n i t i a l l y , a s e n s i t i v e and r e p r o d u c i b l e method o f d e t e c t i o n had t o be d e v e l o p e d . A h i g h performance l i q u i d chromatographic (HPLC) procedure was found t o be a v e r y s e n s i t i v e and e x a c t i n g way o f measuring these a n t i o x i d a n t s a l l o w i n g f o r d e t e c t i o n o f as l i t t l e as 0.1 ug/ml o f a n t i o x i d a n t . Standard c u r v e s were e s t a b l i s h e d r e l a t i n g c o n c e n t r a t i o n o f a n t i o x i d a n t t o peak h e i g h t f o r both BHA and BHT and the r e s u l t s were found t o be b o t h l i n e a r and r e p r o d u c i b l e . A t y p i c a l chromatogram i s shown f o r BHT ( F i g u r e 6) and BHA ( F i g u r e 7) w i t h absorbance b e i n g measured a t * = 280 nm. m

a

x

Samples o f mammary g l a n d , serum and l i v e r from 50 d a y - o l d female Sprague-Dawley r a t s consuming n o n - a n t i o x i d a n t - c o n t a i n i n g s e m i p u r i f i e d d i e t s were o b t a i n e d . To each o f these was added a known amount o f an a n t i o x i d a n t and an e x t r a c t i o n procedure d e v e l o p e d . P r i o r to t h i s time, no simple method has been r e p o r t e d f o r d e t e r m i n i n g t i s s u e conc e n t r a t i o n s o f these compounds, s h o r t o f steam d i s t i l l a t i o n s (37, 38). A m o d i f i c a t i o n o f the B l i g h and Dyer (39) method f o r e x t r a c t i n g l i p i d s was u t i l i z e d f o r the f i r s t s t e p . D e t a i l s o f t h i s procedure are r e p o r t e d i n Terao ^ t a^. ( 4 0 ) . A b r i e f summary i s g i v e n f o r BHA from mammary g l a n d i n F i g u r e 8 (the same procedure i s used f o r BHT but v a r i e s s l i g h t l y f o r serum and l i v e r [ 4 0 ] ) . When s t a n d a r d c u r v e s f o r each o f these compounds were p l o t t e d f o l l o w i n g e x t r a c t i o n , i t was found t h a t average r e c o v e r i e s f o r t h r e e known amounts o f a n t i o x i d a n t s

Publication Date: May 6, 1985 | doi: 10.1021/bk-1985-0277.ch011

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i n samples o f mammary t i s s u e were 98.7% and 99.7% f o r BHT ( F i g u r e 9) and BHA ( F i g u r e 10), r e s p e c t i v e l y . The s t a n d a r d c u r v e s i n d i c a t e d a good c o r r e l a t i o n between peak h e i g h t and a n t i o x i d a n t c o n c e n t r a t i o n , and i t was f e l t t h a t an a c c u r a t e and r e p r o d u c i b l e method f o r measuri n g t i s s u e and serum c o n c e n t r a t i o n s had been d e f i n e d . The a c t u a l experiments began by f e e d i n g female Sprague-Dawley r a t s 0.3% BHT i n a low f a t d i e t f o r v a r i o u s p e r i o d s o f time. At t h e end o f the f e e d i n g p e r i o d , the animals were e u t h a n i z e d i n a chamb e r , mammary t i s s u e removed, p l a c e d i n an i c e b a t h , and trimmed f r e e o f extraneous f a t and lymph g l a n d s . The t i s s u e was e x t r a c t e d and a n a l y z e d by HPLC as d e s c r i b e d . I t was found t h a t BHT i s not r e t a i n e d i n any d e t e c t a b l e amount (< 0.1 ug/ml) i n the serum o r serum l i p i d s o f animals f e d t h e a n t i o x i d a n t f o r two o r t h r e e weeks, o r f o r seventeen weeks. The t o t a l mean l i p i d c o n t e n t i n the serum o f s i x animals f e d BHT f o r two and t h r e e weeks was found to be 2.0 ± 0.7 mg/ml and 1.7 ± 0.1 mg/ml, respectively. Co-chromatograms o f a BHT s t a n d a r d and e x t r a c t s o f l i v e r and mammary t i s s u e c o n f i r m e d t h e presence o f BHT i n b o t h ( F i g u r e 6; mammary, l i v e r not shown). Other than the p r e s e n c e o f the BHT peak,

Is

I 0

1 2

I

i 4

I

I 6

I

I 8

I

I 10

1

1 12

1

L 14

F i g u r e 6. HPLC a n a l y s i s o f mammary t i s s u e e x t r a c t s . (A) Rat f e d low f a t d i e t c o n t a i n i n g 0.3% BHT f o r 2 weeks. (B) C o n t r o l r a t f e d low f a t d i e t o n l y . * The arrow shows t h e p o s i t i o n o f t h e BHT peak.

Publication Date: May 6, 1985 | doi: 10.1021/bk-1985-0277.ch011

144

Figure a diet

XENOBIOTIC M E T A B O L I S M : NUTRITIONAL EFFECTS

7. HPLC a n a l y s i s o f mammary t i s s u e e x t r a c t s from r a t s f e d c o n t a i n i n g b u t y l a t e d h y d r o x y a n i s o l e (BHA).

t h e r e were no d i f f e r e n c e s i n the chromatograms between the c o n t r o l and e x p e r i m e n t a l . Animals f e d BHT f o r two weeks had g r e a t e r c o n c e n t r a t i o n s (0.64 ± 0.26 ug/g) than those f e d the a n t i o x i d a n t f o r t h r e e weeks (0.35 ± 0.09 ug/g o f 1 i v e r ) . BHT l e v e l s found i n the l i v e r l i p i d (33.3 ± 9.7 ug/g) o f animals f e d the a n t i o x i d a n t f o r two weeks was 15.22 ± 2.10 ug/g l i p i d compared t o t h e amount from l i v e r s o f animals on BHT f o r t h r e e weeks o f 10.80 ± 2.71 ug/g l i p i d . BHT l e v e l s found i n mammary t i s s u e and mammary l i p i d a r e shown i n T a b l e V. A h i g h c o n c e n t r a t i o n o f BHT was found i n mammary t i s s u e , a l o n g w i t h s e v e r a l s u s p e c t e d m e t a b o l i t e peaks. The BHT peak was c o l l e c t e d , a n a l y z e d by gas chromatography/mass s p e c t r o s c o p y and conf i r m e d by comparison w i t h an a u t h e n t i c sample.

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Modulation of Mammary Tumor Incidence

KINGETAL.

145

TISSUE

MAMMARY

—

MINCED

—

WEIGH T H E AMOUNTS ( 1 G )

• B L I G H & DYER SOLVENT (MEOH:CHC1 :H 0 = 2:1:0.8) 7.6 ML 3

2

" M I X I N G WITH VORTEX MIXER FOR 1 M I N -HOMOGENIZE WITH POLYTRON HOMOGENIZER FOR 1 MIN. -WASHED WITH B & D SOLVENT

I N AN I C E BATH

7.0 ML

- I N C U B A T E ^ H E A T I N G B L O C K , AT 40'C FOR 1 HOUR -ADD C H C I 3 -ADD H 0

2.0 ML 2.0 ML

2

- M I X I N G WITH VORTEX M I X E R FOR 1 M I N

Publication Date: May 6, 1985 | doi: 10.1021/bk-1985-0277.ch011

- C E N T R I F U G E AT 2500 RPM FOR 10 M I N

LOWER LAYER~| (CHC1 ) 7

I UPPER L A Y E R ] (ME0H-H 0)

REEXTRACTIQN

2

-ADD C H C I 3

2.0 ML

- M I X I N G 1 M I N (VORTEX M I X E R ) - C E N T R I F U G E AT 2500 RPM FOR 10 M I N

1 LOWER LAYER

UPPER

LAYER

-EVAPORATE BY No GAS AT 35°

U0°C

-DRY BY D E S S I C A T O R FOR 2 - 5 HR. - E X T R A C T BY ME0H -EVAPORATE BY N

2

1 ML ( 3 T I M E S ) GAS AT 35°

-ADD 1 ML M O B I L E PHASE HPLC

F i g u r e 8. E x t r a c t i o n p r o c e d u r e used i n t h e d e t e r m i n a t i o n o f BHA i n mammary g l a n d i n r a t s f e d a d i e t c o n t a i n i n g t h i s a n t i o x i d a n t .

BHT l e v e l s i n mammary t i s s u e and mammary l i p i d s o f a n i m a l s p l a c e d on d i e t a r y BHT a t d i f f e r e n t ages a r e shown i n T a b l e V. Group I a n i m a l s a t 91 days o f age were d i v i d e d i n t o t h r e e groups (10/group) and f e d 0.3% BHT i n a LF d i e t f o r 1, 2 and 3 weeks. Group I I a n i m a l s were f e d BHT as above b e g i n n i n g a t 35 days o f age, but f o r 2 o r 17 weeks. BHT was s i g n i f i c a n t l y i n c r e a s e d i n mammary t i s s u e o f Group I fed t h i s a n t i o x i d a n t f o r 2 weeks compared to 1 week, and t h i s i n c r e a s e was seen on a p e r t i s s u e weight o r a p e r l i p i d weight b a s i s . In the Group I I a n i m a l s , BHT l e v e l s were s i g n i f i c a n t l y lower than those i n Group I animals f e d BHT f o r 2 weeks i n d i c a t i n g a d e f i n i t e d i f f e r e n c e i n t i s s u e uptake i n a n i m a l s o f d i f f e r e n t ages. One f a c t o r t h a t might be r e s p o n s i b l e i s t h a t the whole mammary t i s s u e weight i n younger r a t s (Group I I ) was o n l y about o n e - h a l f (1.10 ± 0.19 g) t h a t of the o l d e r r a t s i n Group I (2.45 ± 0.67 g ) .

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146

y

=

80.79x + 1.59

Publication Date: May 6, 1985 | doi: 10.1021/bk-1985-0277.ch011

-.b

A

i

i

0

i

1 1 0.5

i

i

i

I i

i

1.0

i

i

1 i i 1.5

concentration of BHT (pg/ml) F i g u r e 9. Standard curve f o r a n a l y s i s o f BHT. S e n s i t i v i t y i n c r e a s e d 8 times h i g h e r f o r the c o n c e n t r a t i o n range o f 0.1 t o 1.0 ug/ml o f BHT. d e g r e s s i o n l i n e y = mx + b, where y = peak h e i g h t and x = c o n c e n t r a t i o n o f BHT. ^ C o r r e l a t i o n c o e f f i c i e n t .

Y = 0.9995 y = 190.82x-2.667

0.2

Figure

10.

0.4 0.6 0.8 [BHA] ug/ml

Standard curve

1.0

f o r the d e t e r m i n a t i o n

o f BHA by HPLC.

11.

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Modulation of Mammary Tumor Incidence

T3 TJ