Xenobiotics in Foods and Feeds 9780841208094, 9780841210622, 0-8412-0809-3

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Xenobiotics i n Foods and Feeds John W. Finley, EDITOR

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.fw001

University of Iowa Hospitals Daniel E. Schwass, EDITOR U.S. Department of Agriculture

Based on a symposium sponsored by the Division of Agricultural and F o o d Chemistry at the 184th Meeting of the American Chemical Society, Kansas City, Missouri, September 12-17, 1982

ACS

SYMPOSIUM AMERICAN

CHEMICAL

WASHINGTON, D.C.

SERIES 234 SOCIETY 1983

Library of Congress Cataloging in Publication Data

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.fw001

Xenobiotics in foods and feeds. (ACS symposium series, ISSN 0097-6156; 234) "Based on a symposium sponsored by the Division of Agricultural and Food Chemistry at the 184th Meeting of the American Chemical Society, Kansas City, Missouri, September 12-17, 1982." Includes bibliographies and index. 1. Xenobiotics—Addresses, essays, lectures. 2. Food contamination—Addresses, essays, lectures. 3. Food—Analysis—Addresses, essays, lectures. 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 (184th: 1982: Kansas City, Mo.) V. Series. TX571.X45X45 1983 ISBN 0-8412-0809-3

615.9'54

83-15685

Copyright © 1983 American Chemical Society All Rights Reserved. The appearance of the code at the bottom of the first page of each article in this volume indicates the copyright owner's consent that reprographic copies of the article 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. 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 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. P R I N T E D IN THE U N I T E D S T A T E S OF A M E R I C A

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.fw001

ACS Symposium Series M . Joan Comstock, Series

Editor

Advisory Board David L. Allara

Robert Ory

Robert Baker

Geoffrey D. Parfitt

Donald D. Dollberg

Theodore Provder

Brian M . Harney

Charles N. Satterfield

W. Jeffrey Howe

Dennis Schuetzle

Herbert D. Kaesz

Davis L. Temple, Jr.

Marvin Margoshes

Charles S. Tuesday

Donald E . Moreland

C. Grant Willson

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.fw001

FOREWORD T h e A C S S Y M P O S I U M S E R I E S was f o u n d e d i n 1 9 7 4 to p r o v i d e a m e d i u m f o r p u b l i s h i n g symposia q u i c k l y i n b o o k form. T h e f o r m a t of the Series parallels that of the c o n t i n u i n g A D V A N C E S I N C H E M I S T R Y S E R I E S except that i n o r d e r to save time the papers are n o t typeset b u t are r e p r o d u c e d as t h e y are subm i t t e d b y the authors i n camera-ready form. Papers are rev i e w e d u n d e r t h e supervision of the E d i t o r s w i t h t h e assistance of the Series A d v i s o r y B o a r d a n d are selected to m a i n t a i n the integrity of the symposia; however, v e r b a t i m r e p r o d u c t i o n s of p r e v i o u s l y p u b l i s h e d p a p e r s a r e n o t accepted. B o t h reviews a n d reports of r e s e a r c h are a c c e p t a b l e since s y m p o s i a m a y e m b r a c e b o t h types o f presentation.

PREFACE B Y D É F I N I T I O N , A R E C O M P O U N D S that are foreign, but not n e c e s s a r i l y harmful, to a g i v e n living system. F o r this s y m p o s i u m , w e have f o c u s e d o n b i o l o g i c a l s y s t e m s that affect h u m a n s o r animals. T h i s topic represents a m i l d but timely departure f r o m the traditional areas presented i n s y m p o s i a b y the A g r i c u l t u r e a n d F o o d D i v i s i o n . A l t h o u g h considerable d i s c u s s i o n o f various types o f xenobiotic c o m p o u n d s h a s b e e n i n c l u d e d in m a n y s y m p o s i a a n d general sessions, the current effort is the first to e m p h a s i z e a variety o f potentially toxic materials. In the s y m p o s i u m o n w h i c h this b o o k is based, the authors c o v e r a wide variety o f c o m p o u n d s f o u n d in the f o o d chain. T h e s e c o m p o u n d s m a y have plant or m i c r o b i o l o g i c a l o r i g i n o r m a y result f r o m changes c a u s e d b y f o o d storage, processing, o r final preparation. C u r r e n t l y there is high public interest i n the nutritional value a n d safety o f foods. In a d d i t i o n to p r o v i d i n g a source o f essential nutrients, h u m a n f o o d c o n s u m p t i o n c a n represent a delightful social experience a n d contribute t o sensory satisfaction. O r g a n o l e p t i c research a n d c u l i n a r y arts h a v e taught us to p r o d u c e f o o d s with extraordinary c o n v e n i e n c e a n d p a l a t a b i l i t y . M i c r o b i o l o g i c a l r e s e a r c h h a s h e l p e d u s prevent age-old public health p r o b l e m s b y ensuring sanitary handling o f f o o d s throughout p r o d u c t i o n a n d processing. N u t r i t i o n i s t s have established requirements and e n s u r e d the nutrient quality o f foods, a n d w e n o w have the opportunity to c o n s u m e a nutritionally adequate diet. T h e o v e r a l l i m p r o v e m e n t in f o o d handling i n the last 100 years n o w challenges us with n e w considerations o f what constitutes a safe diet. T h e influence o f xenobiotics o n these c o n s i d e r a t i o n s is not c l e a r l y u n d e r s t o o d . F o o d toxicology, particularly as related t o long-term effects o f low-level e x p o s u r e to toxic materials, is n o w e m e r g i n g as a b r a n c h o f f o o d science. M o s t o f the f o o d s w e c o n s u m e are beneficial; s o m e foods, however, m a y h a v e risks. R a t i o n a l assessment o f these risks a n d benefits is a n e x t r e m e l y difficult area o f research and, t o o frequently, a n e m o t i o n a l issue. T h i s v o l u m e presents a n objective, state-of-the-art d i s c u s s i o n o f xen o b i o t i c c o m p o u n d s that c a n exist i n the diet a n d includes s o m e o f the

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.pr001

u^CENOBIOTICS,

vii

a p p r o a c h e s available to m i n i m i z e the o c c u r r e n c e o f these c o m p o u n d s . W e a r e v e r y grateful f o r assistance a n d w o u l d like to thank the A g ricultural D i v i s i o n o f the C i b a - G e i g y C o r p o r a t i o n a n d Pfizer, Inc. f o r their generous support w h i c h e n a b l e d several speakers to attend.

JOHN W. FINLEY

University of Iowa Hospitals Department of Pediatrics Iowa City, Iowa Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.pr001

DANIEL E. SCHWASS

U.S. Department of Agriculture Berkeley, California June 16, 1983

viii

1 Systematic Toxicity Testing for Xenobiotics in Foods

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch001

MORRIS M. JOSELOW University of Medicine of New Jersey, Newark, NJ 07103

Substances that are not natural to a food, xenobiotics, may become a part of the food by several routes. They may be deliberately added as food or color additives; they may result indirectly from migration of additives from wrappings into the food; they may also result from the incorporation of environmental pollutants into the food while that food is growing or maturing. All such xenobiotics inevitably raise questions of safety. This, of course, does not infer that a l l natural foods are safe (1). We know this to be untrue--but it does recognize that anything added to foods will be regarded with suspicion until adequate toxicity testing has established its safety--a concept that now applies to almost a l l foods and drugs introduced into commerce. As might be expected, there i s a large interface between toxicity testing and legal requirements. Some formal definitions, originally proposed by the National Academy of Sciences, are in order. Toxicity is defined as the capacity of a substance to cause injury, in a very broad sense, and includes injury to any mechanism or tissue of the body, as well as i r r i t a t i o n , behavioral changes, or mental disturbances. Hazard, as used, for example, in defining a hazardous substance, involves a probability concept, like so much else in toxicology. It is an estimate of the probability or likelihood that a substance may cause injury. The converse of this i s the meaning reserved for the term safety, namely, the probability (or practical certainty) that injury will not result when a substance i s used in a particular manner and quantity (2). The use aspect is a major qualification that inheres in these definitions. Any substance can cause injury i f high enough concentrations are administered. The evaluation of safety or hazard must therefore take into account the conditions of use or possible predictable misuse of the substance. Toxicity testing has become a major concern nationally because of the extensive legislation that now mandates such testing (Food, Drug, and Cosmetic Act; Hazardous Substances Act; Occupational Safety and Health Act; Toxic Substances Control Act) as well as i n ternationally, because of the need for mutually acceptable products as a prerequisite to economic cooperation in the marketing of foods and chemicals. 0097 -6156/83/0234-0001 $06.00/0 © 1983 American Chemical Society

2

X E N O B I O T I C S IN F O O D S A N D

FEEDS

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch001

In no o t h e r a r e a has t o x i c i t y t e s t i n g assumed g r e a t e r i m p o r t a n c e t h a n w i t h f o o d s , f o r t h e o b v i o u s r e a s o n t h a t f o o d s r e p r e s e n t pot e n t i a l l y t h e g r e a t e s t p o p u l a t i o n i m p a c t , and t h e r e f o r e t h e need for greatest surveillance. B e f o r e p r o c e e d i n g w i t h c o n s i d e r a t i o n o f some o f t h e f o r m a l t e s t p r o c e d u r e s t h a t have been d e v e l o p e d and a r e now i n u s e , we s h o u l d recognize a fundamental flaw i n the s c i e n t i f i c b a s i s f o r a l l safety e v a l u a t i o n s . A l l t e s t s t o evaluate safety are designed to demonstrate a negative, the p r o b a b i l i t y t h a t i n j u r y w i l l not r e s u l t , w h i c h , by d e f i n i t i o n and l o g i c , c a n n o t be p r o v e n . We may sometimes f e e l , a f t e r h a v i n g p e r f o r m e d a s u f f i c i e n t number of t e s t s o f a s u f f i c i e n t v a r i e t y over a s u f f i c i e n t length o f time, t h a t s a f e t y has been f u l l y d e m o n s t r a t e d . But i t o n l y t a k e s a t h a i i d o m i d e - t y p e d i s a s t e r , w i t h t h e a p p e a r a n c e o f a new u n e x p e c t ed e f f e c t , t o r e m i n d u s t h a t a b s o l u t e s a f e t y -- t h e g u a r a n t e e d a b s e n c e o f any harm -- i s an i l l u s i o n t h a t can n o t be u n e q u i v o c a l l y e s t a b l i s h e d , u n l e s s we c o u l d t e s t a l l human b e i n g s o v e r an i n f i n i t e p e r i o d o f time, an o b v i o u s l y impossible task. Faced with t h i s c o n f l i c t between w i s h and r e a l i t y , we have come t o r e l y on " s c i e n t i f i c j u d g m e n t " -- a much o v e r u s e d and a b u s e d p h r a s e t h a t s h o u l d a l w a y s be s u s p e c t -- t o make p r e d i c t i o n s a b o u t s a f e t y t h a t we t h e n c l a i m a r e beyond r e a s o n a b l e d o u b t , o r t h a t t h e r e i s no unr e a s o n a b l e r i s k " , a s r e q u i r e d by t h e T o x i c S u b s t a n c e s C o n t r o l A c t . Of n e c e s s i t y , heavy r e l i a n c e i n t o x i c i t y t e s t i n g must be p l a c e d on t h e use o f a n i m a l s a s s u r r o g a t e s o r s u b s t i t u t e s f o r humans. T h i s r e q u i r e m e n t has imposed some q u a l i f i c a t i o n s -- a t t i m e s s e v e r e -- i n t h e i n t e r p r e t a t i o n o f r e s u l t s . Choice o f S p e c i e s o f T e s t Animal In d e c i d i n g o n t h e animal t o be u s e d , t o x i c o l o g i s t s a t t e m p t t o f o l l o w a b a s i c g u i d e l i n e : w h e r e v e r p o s s i b l e , use a s p e c i e s t h a t b i o l o g i c a l l y handles the m a t e r i a l under study a s s i m i l a r l y a s p o s s i b l e t o man; and, i n d e c i d i n g s i m i l a r i t y t o man, c o n s i d e r a t i o n s o f m e t a b o l i s m , a b s o r p t i o n , e x c r e t i o n , and o t h e r p h y s i o l o g i c a l p a r a m e t e r s s h o u l d be t a k e n i n t o a c c o u n t . T h i s g u i d e l i n e , w h i c h w o u l d seem t o be s o f u n d a m e n t a l , n e v e r t h e l e s s , p o s e s a dilemma. The t o x i c o l o g i s t would l i k e t o use s p e c i e s t h a t i s a s s e n s i t i v e t o t h e i n j u r y e x p e c t e d f o r t h e substance under study, a s p e c i e s t h a t would r e a c t p h y s i o l o g i c a l l y l i k e humans, o r be even more s e n s i t i v e . But t h e s t a t e o f t h e a r t i n t o x i c o l o g y i s n o t s u f f i c i e n t l y developed t o permit p r e d i c t i o n s as to which species of animals are most s i m i l a r t o man i n t h e i r r e s p o n s e s t o c h e m i c a l c h a l l e n g e s . In p r a c t i c e , t h e c h o i c e o f a s p e c i e s i s o f t e n d i c t a t e d more by p r a c t i c a l c o n s i d e r a t i o n s t h a n by s i m i l a r i t y o f r e s p o n s e . C o n -

1.

JOSELOW

Systematic

Toxicity

Testing in Foods

3

venience, ease o f h a n d l i n g , a v a i l a b i l i t y o f stock, housing f a c i l i t i e s , c o s t o f m a i n t e n a n c e , and p r e c e d e n c e o f use -- a l l o f t h e s e e n t e r i n t o a judgment o f t h e c h o i c e o f s p e c i e s . A l b i n o r a t s and dogs have been most f r e q u e n t l y u s e d , b e c a u s e t h e y a r e t h e most e a s i l y a v a i l a b l e a n i m a l s and have a l o n g h i s t o r y o f u s e .

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch001

The f a c t t h a t r a t s and dogs a r e most w i d e l y u s e d i s a l s o s e l f perpetuating. I n any s t u d y o f a new c h e m i c a l , t h e o b v i o u s f i r s t c h o i c e w o u l d be an animal s p e c i e s whose r e a c t i o n s a r e w e l l - k n o w n and documented. A l s o , t h e p r a c t i c a l p r o b l e m o f o b t a i n i n g s u i t a b l e a n i m a l s i s m i n i m i z e d by t h e use o f t h o s e f o r w h i c h p r e v i o u s demands have c r e a t e d a d e q u a t e , d e p e n d a b l e , a n d i n e x p e n s i v e s o u r c e s of supply. F u r t h e r m o r e , some f e d e r a l r e g u l a t i o n s have recommended and t h e r e by a l m o s t mandated t h e use o f r a t s and d o g s , p a r t i c u l a r l y , f o r l o n g - t e r m t o x i c i t y s t u d i e s o f f o o d a d d i t i v e s and p e s t i c i d e s , e v e n t h o u g h t h e r e may be i m p o r t a n t d i f f e r e n c e s i n m e t a b o l i c c a p a b i l i t i e s and p h y s i o l o g i c a l r e s p o n s e s between t h e s e s p e c i e s and man. The r a t i s a p o o r c h o i c e t o e v a l u a t e t h e l i v e r i n j u r y p o t e n t i a l o f a s u b s t a n c e f o r man, b e c a u s e t h e r a t l i v e r i s r e s i s t a n t , a n d r e g e n e r a t e s r a p i d l y . The g u i n e a p i g o r t h e r a b b i t w o u l d be a b e t t e r c h o i c e o f t h i s . The dog does n o t a c e t y l a t e o r d e t o x i f y aroma t i c a m i n e s . The monkey and t h e g u i n e a p i g r e q u i r e exogenous a s c o r b i c a c i d , w h i l e t h e r a t does n o t , w h i c h makes t h e r a t u n s u i t a b l e f o r t h e d e m o n s t r a t i o n o f a n a s c o r b i c and d e f i c i e n c y . For long-term s t u d i e s , a s p e c i e s having a r e l a t i v e l y s h o r t l i f e span p e r m i t s d e t e r m i n i n g t h e e f f e c t s o f a c h e m i c a l o v e r c o u r s e o f a l i f e t i m e . The l a t t e r r e q u i r e m e n t , i n a p r a c t i c a l s e n s e , l i m i t s l i f e - t i m e s t u d i e s t o r a t s or mice o r hamsters. But h e r e t o o , some b a s i c d i f f i c u l t i e s can p r e s e n t t h e m s e l v e s . F o r a s s e s s i n g t h e p o s s i b i l i t y o f c a n c e r -- t h e main o b j e c t i v e i n l o n g t e r m t e s t s -- we r e l y o n t h e use o f h e a l t h y , d i s e a s e - f r e e i n b r e d mice, which represent c h a r a c t e r i s t i c s t h a t are h a r d l y r e p r e s e n t a t i v e o f a h e t e r o g e n e o u s human p o p u l a t i o n a t r i s k . T h i s d i f f e r e n c e o n l y c o m p l i c a t e s known d i f f e r e n c e s i n m e t a b o l i c r e s p o n s e s ; e.g., m i c e can n o t d e c a r b o x y l a t e and e l i m i n a t e some c a r c i n o g e n i c h y d r o c a r b o n s t h a t humans can r e a d i l y " d e t o x i f y " . F o r p r e l i m i n a r y s t u d i e s and s c r e e n i n g , r a t s , m i c e , r a b b i t s , o r g u i n e a p i g s a r e s e l e c t e d f o r economy, e a s e o f t r e a t m e n t , and, t o a l a r g e e x t e n t , a c c o r d i n g t o t h e r e p o r t e d work o f o t h e r s who have done s i m i l a r s t u d i e s . F o r t h e c h r o n i c s t u d i e s r e q u i r e d by some F e d e r a l r e g u l a t i o n s , t h e s p e c i e s s p e c i f i e d by t h e a g e n c i e s a r e u s e d , u s u a l l y r a t s and b e a g l e d o g s . U n d e r t h e F e d e r a l H a z a r d o u s S u b s t a n c e A c t ( 3_ ) f o r e x a m p l e , t h e d e f i n i t i o n s o f t o x i c , h i g h l y t o x i c , and n o n - t o x i c

4

X E N O B I O T I C S IN F O O D S A N D

FEEDS

a r e b a s e d i n p a r t on r e s p o n s e s o f r a t s t o o r a l o r i n h a l a t i o n exp o s u r e s . T h a t recommendation has made i t a l m o s t m a n d a t o r y t o use r a t s and f o l l o w t h e p r e s c r i b e d p r o t o c o l s . In summary, t h o u g h we o u g h t t o use a s p e c i e s r e a c t i n g q u a l i t a t i v e l y and q u a n t i t a t i v e l y most l i k e man, what we a c t u a l l y use most o f t e n i s the s p e c i e s f o r which h e a l t h y commerical s t o c k s , reasona b l y p r i c e d and r e a s o n a b l y c o n s t a n t , a r e a v a i l a b l e .

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch001

A d m i n i s t r a t i o n o f Dose The r o u t e c h o s e n f o r a d m i n i s t r a t i o n o f t h e s u b s t a n c e t o t h e t e s t animal and t h e manner o f i t s a d m i n i s t r a t i o n s h o u l d be t h e same a s t h a t by w h i c h man w i l l be e x p o s e d . I n a c u t e t o x i c i t y t e s t i n g , however, t h i s i s a p r a c t i c e t h a t i s i n t e n t i o n a l l y v i o l a t e d , s i n c e a s i n g l e m a s s i v e dose i s u s u a l l y a d m i n i s t e r e d by i n t u b a t i o n o r gavage. T h e r e w i l l a l w a y s be some d i f f e r e n c e s between t h e e f f e c t s o b s e r v ed i n a n i m a l t e s t s , and a c t u a l human e x p e r i e n c e . No i n t u b a t e d o r a l d o s e can be r e l i e d upon t o be a sound model o f t h e e f f e c t o f a c h e m i c a l i n t h e d i e t , even i f i t i s r e p e a t e d day a f t e r day. T h e s i n g l e d o s e w i l l o f n e c e s s i t y y i e l d a peak c o n c e n t r a t i o n i n body f l u i d s h i g h e r t h a n what w o u l d r e s u l t from t h e s l o w a b s o r p t i o n o f a c h e m i c a l d u r i n g s l o w d i g e s t i o n o f f o o d . I n t u b a t i o n a l s o has a g r e a t e r c h a n c e o f b e i n g i n j u r i o u s , e i t h e r by t h e g r e a t e r m a g n i tude o f t h e peak b l o o d c o n c e n t r a t i o n o r by o v e r w h e l m i n g a metab o l i c pathway t h a t c o u l d i n normal c i r c u m s t a n c e s h a n d l e a l o w e r c o n c e n t r a t i o n o f the t o x i c substances. Dose L e v e l s and S a f e t y F a c t o r s A b a s i c assumption underlying t o x i c i t y t e s t i n g i s t h a t responses a r e d o s e - r e l a t e d , and t h a t , i n t e s t i n g , s e v e r a l d o s e s s h o u l d be administered to e l i c i t graded responses. F u r t h e r m o r e , t h e r e must a l s o be some dose below w h i c h no r e s p o n s e w i l l be shown ( o r , more p r e c i s e l y , be i n c a p a b l e o f d e t e c t i o n ) . T h i s dose has been v a r i o u s l y t e r m e d t h e " b i o l o g i c a l l y i n s i g n i f i c a n t d o s e " o r t h e "NOEL" (No O b s e r v e d E f f e c t L e v e l ) , and i t s d e t e r m i n a t i o n i s one o f t h e primary o b j e c t i v e s o f t o x i c i t y t e s t i n g . The term " a d v e r s e e f f e c t " i s n o t e a s i l y i n t e r p r é t a b l e . A p h y s i o l o g i c a l r e s p o n s e t o a s t r e s s t h a t i s r e a d i l y r e v e r s i b l e , such a s a change i n enzyme c o n c e n t r a t i o n s , m i g h t n o t n e c e s s a r i l y be a n a d v e r s e e f f e c t , p a r t i c u l a r l y i f no damage i s d e t e c t a b l e . The a b i l i t y o f t o x i c i t y t e s t i n g t o s e t s a f e l e v e l s i s i t s most i m p o r t a n t p r a c t i c a l f u n c t i o n . The p r o c e d u r e has been d e v e l o p e d most r i g o r o u s l y i n t h e f i e l d o f f o o d and c o l o r a d d i t i v e t e s t i n g -- c h e m i c a l s added t o f o o d s -- where a s e r i e s o f s p e c i a l s t i p u l a -

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch001

1.

JOSELOW

Systematic

Toxicity

Testing in Foods

5

tions that permit estimates o f " t o x i c o l o g i c a l l y i n s i g n i f i c a n t l e v e l s " o f c h e m i c a l s i n f o o d s , have been p r o p o s e d . I n g e n e r a l , a " s a f e " l e v e l o f a s u b s t a n c e i s a r b i t r a r i l y s e t by a p p l y i n g a s a f e t y f a c t o r t o the h i g h e s t intake t h a t i s found not t o i n j u r e experimental animals exposed f o r extended p e r i o d s ; i . e . , over a l i f e t i m e . The "safe" level i s f r e q u e n t l y expressed as l/100th o f t h e e x p e r i m e n t a l l y d e t e r m i n e d h i g h e s t No O b s e r v e d E f f e c t L e v e l . T h i s r a t i o , 1:100, i s d e r i v e d by a p p l y i n g a f a c t o r o f 10 f o r ext r a p o l a t i o n o f t h e f i n d i n g s o f a n i m a l s t o man, a n d a n o t h e r f a c t o r o f 10 t o a c c o u n t f o r v a r i a t i o n s i n s u s c e p t i b i l i t i e s among p e o p l e . A r a t i o o f 100 i s t h o u g h t t o p r o v i d e a c o n s e r v a t i v e e s t i m a t e o f t h e s a f e t y f a c t o r needed t o a f f o r d a d e q u a t e p r o t e c t i o n , even t o p e r s o n s whose d i e t a r y p a t t e r n s o r i n d i v i d u a l s u s c e p t i b i l i t i e s m i g h t be u n u s u a l . A n d t h i s p r o c e d u r e s has been s o w i d e l y a d o p t e d by n a t i o n a l a n d i n t e r n a t i o n a l b o d i e s c o n c e r n e d w i t h s a f e t y t h a t the term " s a f e l e v e l " , a s a p p l i e d t o chemicals i n f o o d , has a l s o become a l a r g e e x t e n t a p p l i c a b l e t o c h e m i c a l s a n d h a z a r d o u s s u b s t a n c e s t h a t impinge on man f r o m w h a t e v e r s o u r c e . The c o n c e p t t h a t t h e r e must be some dose l e v e l below which an a d v e r s e e f f e c t w i l l n o t a p p e a r has n o t met w i t h u n i v e r s a l a c c e p t a n c e , p a r t i c u l a r l y w i t h r e g a r d t o t o x i c a g e n t s t h a t have l o n g t e r m , i r r e v e r s i b l e e f f e c t s , such a s i o n i z i n g r a d i a t i o n o r c a r c i n o g e n i c s u b s t a n c e s . Some t o x i c o l o g i s t s have p r o p o s e d t h a t a d e q u a t e t e s t i n g c a n s e t s a f e l i m i t s even f o r c a r c i n o g e n i c a g e n t s ; o t h e r s c h a l l e n g e t h i s b e l i e f , and a r g u e t h a t i t i s i m p o s s i b l e t o p r e d i c t s a f e l e v e l s f o r c a r c i n o g e n s by t a k i n g an a r b i t r a r y f r a c t i o n o f t h e l o w e s t No O b s e r v e d E f f e c t a n i m a l dose i n any p a r t i c u l a r e x p e r i m e n t a l s i t u a t i o n . S h a r p d i s t i n c t i o n s have a l s o been made between r e v e r s i b l e and i r r e v e r s i b l e e f f e c t s . F o r a d d i t i v e s t h a t can i n d u c e r e v e r s i b l e t o x i c e f f e c t s , t h r e s h o l d l e v e l s below which human e x p o s u r e w o u l d be s a f e c a n be r e a s o n a b l y d e t e r m i n e d . Howe v e r , f o r c h e m i c a l s i n d u c i n g i r r e v e r s i b l e and p o s s i b l y c u m u l a t i v e e f f e c t s , such t h r e s h o l d s c a n n o t be d e t e r m i n e d ; and a z e r o t o l e r a n c e has t o be s e t . I f a z e r o t o l e r a n c e i s m a n d a t e d , t h e n t h e r e i s no need f o r t o x i c i t y t e s t i n g . O n l y t h e t e c h n i q u e s o f a n a l y t i c a l c h e m i s t r y need be a p p l i e d t o d e t e r m i n e w h e t h e r a s u b s t a n c e i s p r e s e n t o r n o t , and t h e r e f o r e , w h e t h e r i t s u s e i s p e r m i s s i b l e o r n o t . Such an a p p r o a c h however, r u n s t h e r i s k o f becoming a r e d u c t i o a d a b s u r dum, a s a n a l y t i c a l t e c h n i q u e s become more r e f i n e d and s e n s i t i v e . D e s i g n s and O b j e c t i v e s f o r Formal T e s t i n g The movement toward s y s t e m a t i c f o r m a l i z e d t o x i c i t y t e s t i n g b e g a n , as m i g h t be e x p e c t e d , w i t h t h e p r o b l e m o f c h e m i c a l s i n f o o d s i n t h e 1940's. A Food and Drug A d m i n i s t r a t i o n r e p o r t p u b l i s h e d i n 1943 ( 4 ) o f f e r e d some g e n e r a l p r o t o c o l s . As a d d i t i o n a l t o x i c e f f e c t s ' - - u n s u s p e c t e d a t t h a t t i m e -- became known, t h e s e r e c -

6

X E N O B I O T I C S IN F O O D S A N D

FEEDS

ommendations were a m p l i f i e d i n d e t a i l and expanded i n s c o p e . T h e r e c e n t l y p u b l i s h e d monograph ( ) o f t h e Bureau o f Goods, Food and Drug A d m i n i s t r a t i o n , e n t i t l e d , " T o x i c o l o g i c a l P r i n c i p l e s " can be r e g a r d e d a s t h e c u l m i n a t i o n o f t h e s e e f f o r t s . I t i s a c o n s i d e r a b l y updated v e r s i o n o f the o r i g i n a l recommendations, and i s s i n e qua non f o r t h o s e c o n c e r n e d w i t h t o x i c i t y t e s t i n g .

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch001

I t p r o v i d e s d e t a i l s r e g a r d i n g t e s t r e q u i r e m e n t s and p r o t o c o l s , and r e p r e s e n t s t h e c o n s e n s u s o f j u d g m e n t s o f t h e a g e n c y most c o n cerned with the s a f e t y o f x e n o b i o t i c s i n foods. The o r i g i n a l g u i d e l i n e s recommended o n l y t h r e e b a s i c t y p e s o f t e s t s t o be p e r f o r m e d on l a b o r a t o r y a n i m a l s t o e v a l u a t e s a f e t y . W h i l e o t h e r t e s t s f o r s p e c i f i c t o x i c e f f e c t s have s i n c e been a d d e d , t h e s e b a s i c t e s t s s t i l l r e m a i n t h e backbone o f c u r r e n t t o x i c i t y t e s t i n g procedures. These t e s t s d i f f e r p r i m a r i l y i n t h e i r d u r a t i o n s and o b j e c t i v e s . T e s t s t h a t use o n l y s i n g l e d o s e s o f t h e c h e m i c a l , a d m i n i s t e r e d on one o c c a s i o n , a r e r e f e r r e d t o a s "acute t e s t s " . Longer t e s t s , i n which the chemical i s given a t l e a s t o n c e d a i l y , f o r p e r i o d s o f up t o t h r e e months, a r e commonly r e f e r r e d t o as "sub-acute" o r "sub-chronic" o r "prolonged" t e s t s . S t i l l l e n g t h i e r t e s t s , i n v o l v i n g the a d m i n i s t r a t i o n o f a chemical t o a n i m a l s d a i l y f o r p e r i o d s o f one t o two y e a r s t o s i m u l a t e l i f e time exposure, are r e f e r r e d t o as " c h r o n i c " o r "long term" o r "extended" t o x i c i t y t e s t s . Acute T o x i c i t y Tests The s i n g l e t e s t t h a t i s c o n d u c t e d on p r a c t i c a l l y a l l s u b s t a n c e s of b i o l o g i c i n t e r e s t i s the acute t o x i c i t y t e s t . I t i s undoubte d l y r e s p o n s i b l e f o r t h e g r e a t e s t r i t u a l mass s l a u g h t e r o f a n i mals i n t h i s country. The t e s t r e q u i r e s t h a t t h e a n i m a l s be e x p o s e d t o a r e l a t i v e l y l a r g e d o s e , o n a t l e a s t one o c c a s i o n , t o t h e c h e m i c a l o f i n t e r e s t . The p r i n c i p a l p u r p o s e i s t o d e t e r m i n e t h e l e t h a l i t y o r LD50 f o r t h e c h e m i c a l ( t h e d o s e t h a t w i l l be f a t a l t o 50% o f t h e t e s t population.) A l m o s t a l l a c u t e t o x i c i t y t e s t s a r e done w i t h r a t s o r m i c e , l a r g e ly b e c a u s e o f t h e low c o s t , e a s y a v a i l a b i l i t y , e x p e n d a b i l i t y o f t h e s e a n i m a l s , and t h e f a c t t h a t an a b u n d a n t l i t e r a t u r e e x i s t s f o r t h e s e two s p e c i e s . I n i t i a l l y , t h e c h e m i c a l may be g i v e n t o a s i n g l e s p e c i e s ; a t s e v e r a l dosage l e v e l s , t o p r e - c o n d i t i o n e d g r o u p s o f a n i m a l s , a s p r e s c r i b e d by F e d e r a l r e c o m m e n d a t i o n s . F o l l o w i n g a d m i n i s t r a t i o n o f t h e d o s e s , o b s e r v a t i o n s a r e made o f t h e a n i m a l s f o r p e i o d s r a n g i n g f r o m a few m i n u t e s t o two weeks, t o c o m p l y w i t h t h e r e q u i r e m e n t s o f some r e g u l a t i o n s e.g.; t h e F e d e r a l H a z a r d o u s S u b s t a n c e s A c t . T h e l e t h a l i t y i s d e t e r m i n e d on t h e b a s i s o f d e a t h o c c u r i n g w i t h i n t h e o b s e r v a t i o n p e r i o d (14 days)-

1.

JOSELOW

Systematic

Toxicity

Testing in Foods

7

Subchronic o r Subacute T o x i c i t y T e s t i n g The a c u t e t e s t i s p r i m a r i l y a f a c t - f i n d i n g , e x p l o r a t o r y e x p e r i m e n t t o o b t a i n some i n d i c a t i o n o f t o x i c i t y . Such i n f o r m a t i o n i s u s e d n o t o n l y t o p e r m i t c o m p a r i s o n s among d i f f e r e n t a g e n t s (by compari n g t h e i r L D s q ' S ) , b u t a l s o t o p r o v i d e a more r e f i n e d b a s i s f o r t h e s e l e c t i o n o f dose l e v e l s f o r t h e more p r o l o n g e d t y p e o f s t u d i e s , e x t e n d i n g f o r 3-4 months, t h e s o - c a l l e d " s u b c h r o n i c " o r "subacute animal s t u d i e s .

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch001

These more p r o l o n g e d s t u d i e s a r e d e s i g n e d t o p r o v i d e a d d i t i o n a l i n f o r m a t i o n on t o x i c i t y . They have t h r e e main p u r p o s e s : 1)

T o e s t a b l i s h more n a r r o w l y a maximum t o l e r a t e d d o s e ; i . e . , a dose t h a t w i l l p r o d u c e o v e r t a d v e r s e symptoms b u t n o t death.

2)

T o p r o v i d e an e s t i m a t e o f t h e h i g h e s t dose t h a t w i l l show any e f f e c t ; i . e . , t h e "no e f f e c t " l e v e l .

3)

T o e s t a b l i s h t h e b i o l o g i c a l n a t u r e o f t h e damage p r o d u c e d as r e v e a l e d by c l i n i c a l and p a t h o l o g i c a l e x a m i n a t i o n o f the s a c r i f i c e d animals.

not

Chronic Toxicity I f t h e s u b s t a n c e has been f o u n d t o be s a t i s f a c t o r y , i . e . , nont o x i c , i n t h e s u b c h r o n i c t e s t s , i t may t h e n be s u b j e c t e d t o c h r o n i c , l o n g - t e r m , o r e x t e n d e d t o x i c i t y t e s t s . These t e s t s a r e meant t o s i m u l a t e l i f e t i m e human e x p e r i e n c e , and t h e e x p e r i m e n t a l p r o t o c o l s , t h e r e f o r e , c a l l f o r dosing the animal with the substance, o v e r t h e c o u r s e o f i t s l i f e - t i m e , which means, f o r p r a c t i c a l p u r p o s e s , t h a t an a n i m a l w i t h a s h o r t l i f e span must be u s e d ; i . e . , r a t s or mice. The r a t i o n a l e f o r a c c e p t i n g t h i s i s t h a t i n a s h o r t l i v e d s p e c i e s , p r o g r e s s i v e i n j u r y p r o c e e d s more r a p i d l y , and can be d e t e c t e d more e a s i l y t h a n i n a l o n g - l i v e d s p e c i e s . L i f e t i m e d o s i n g o f a r a t ; i . e . , f o r 30 months, i s c o n s i d e r e d e q u i v a l e n t t o 70 y e a r s e x p o s u r e i n man. The F . D . A . t h u s s p e c i f i e s and a c c e p t s 30 month e x p o s u r e i n r a t s f o r a l i f e t i m e s t u d y . But t h i s r a t i o n a l e does n o t h o l d f o r dogs o r o t h e r a n i m a l s . Two y e a r s f o r a dog i s s t i l l o n l y a f r a c t i o n (20%) o f i t s l i f e t i m e . By t h e t i m e a s u b s t a n c e i s c o n s i d e r e d f o r c h r o n i c t o x i c i t y s t u d i e s , i n f o r m a t i o n w i l l have a l r e a d y been o b t a i n e d r e g a r d i n g t h e n a t u r e o f i t s t o x i c i t y and i t s t o l e r a b l e a s w e l l a s l e t h a l d o s e s . The main p u r p o s e s o f l o n g - t e r m t e s t i n g t h e n a r e t w o f o l d : 1)

To f i n d o r c o n f i r m t h e " N o - E f f e c t " l i f e t i m e dosage

level

X E N O B I O T I C S IN F O O D S A N D F E E D S

8

f o r the a d d i t i v e , i . e . , t h e maximum t h a t can be t a k e n t h a t w i l l n o t p r o d u c e any o b s e r v a b l e a d v e r s e e f f e c t o v e r a 1 i f e time.

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch001

2)

T o d e t e c t any more s i g n i f i c a n t a b n o r m a l i t i e s t h a t may become a p p a r e n t o n l y o v e r t h e c o u r s e o f a l i f e t i m e ; a n d t h e change g e n e r a l l y s o u g h t most commonly i s t h e d e v e l o p ment o f a c a n c e r o f some k i n d .

T h i s i s u n d o u b t e d l y t h e most i m p o r t a n t o b j e c t i v e o f t h e c h r o n i c t e s t -- t o s e a r c h f o r t h e d e v e l o p m e n t o f a c a r c i n o g e n i c e n d r e s u l t . I n f a c t , some t o x i c o l o s i t s b e l i e v e t h a t i f e f f e c t s o t h e r than c a n c e r a r e s o u g h t , t h e s e s h o u l d have become a p p a r e n t i n t h e s h o r t e r s u b c h r o n i c s t u d i e s -- a n d t h e r e would t h e n u s u a l l y be no need t o c a r r y the t e s t beyond t h e s u b c h r o n i c p e r i o d . As m i g h t be e x p e c t e d when c o n d u c t i n g t e s t s w i t h a n i m a l s t h a t w i l l u n a v o i d a b l y v a r y t o some e x t e n t i n t h e i r g e n e t i c make-up, v a r i a t i o n s i n r e s u l t s may be e n c o u n t e r e d , p a r t i c u l a r l y i f t e s t c o n d i t i o n s a r e not c a r e f u l l y c o n t r o l l e d . R e c o g n i t i o n o f t h i s p r o b l e m w h i c h has a c c o u n t e d f o r w i d e d i s p a r i t i e s r e p o r t e d i n t h e l i t e r a t u r e i n t o x i c i t y t e s t f i n d i n g s , h a s l e d F.D.A. and o t h e r s (6) t o s t a n d a r d i z e the r e l e v a n t f a c t o r s i n v o l v e d i n conducting t o x i c i t y t e s t s . T e s t c o n d i t i o n s , i f n o t c o n t r o l l e d , can l e a d t o s p u r i o u s r e s u l t s among d i f f e r e n t l a b o r a t o r i e s , and even w i t h i n t h e same laboratory (7). The s t a n d a r d i z e d f a c t o r s i n c l u d e n o t o n l y t h e c o n d i t i o n s u n d e r w h i c h t h e t e s t s a r e c o n d u c t e d -- and good l a b o r a t o r y p r a c t i c e s a r e now a n added minimum r e q u i r e m e n t -- but a l s o p r e s c r i b e t h e c l i n i c a l t e s t i n g and o b s e r v a t i o n s t h a t must be made on t h e t e s t a n i m a l s d u r i n g and a f t e r the t e s t p e r i o d ( T a b l e s I - I V ) . R e c e n t a d d i t i o n s t o t h e t o x i c i t y t e s t p r o t o c o l s may now r e q u i r e formulized examinations f o r genetic e f f e c t s ( i . e . , teratogenesis, m u t a g e n e s i s , r e p r o d u c t i o n ) a s w e l l a s d e t e r m i n a t i o n o f t h e metabo l i c a l t e r a t i o n s o f t h e a d d i t i v e a n d t h e i r d i s p o s i t i o n . Each o f t h e s e a c t i v i t i e s has become an i m p o r t a n t s u b - b r a n c h o f t o x i c i t y testing. A t y p i c a l manner i n w h i c h a d e c i s i o n on s a f e t y may be made, b a s e d on s e q u e n t i a l t e s t i n g , i s shown i n F i g u r e 1. Such t e s t i n g o b v i o u s l y r e p r e s e n t s an e x t e n s i v e and e x p e n s i v e u n d e r t a k i n g . The s e r v i c e s o f v a r i e d p r o f e s s i o n a l s w i l l be r e q u i r e d : t o x i c o l o g i s t s , p a t h o l o g i s t s , chemists, biochemists, v e t e r i n a r i a n s , s t a t i s t i c i a n s , and a t t o r n e y s ( t o p r e p a r e t h e p e t i t i o n s ) and a h o s t o f o t h e r s u p p o r t ing p e r s o n n e l . From a l l t h e t o x i c i t y d a t a , i f t h e s t u d y works o u t , w i l l emerge a r e a s o n a b l e f i g u r e f o r a "No E f f e c t L e v e l " a s w e l l a s an i n d i c a t i o n

1.

JOSELOW

Systematic

Toxicity

Testing in Foods

TABLE 1 OUTLINE OF GENERAL PROCEDURES*

Animals

No. o f Dose L e v e l s

No. o f A n i m a l s (minimum/group)

ACUTE

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch001

WTrs) r a t s , mice, guinea pigs

Dermal

4 -5

5-10 e a c h , e q u a l n o s . o f e a c h sex

dogs

u s u a l l y t o t a l o f 12

rabbits

2 male & 2 f e m a l e e a c h

INTUBATION 1 - 8 hrs SUBACUTE (90 d a y s )

Dermal

rats

10 male & 10 f e m a l e e a c h

dogs

2 male & 2 f e m a l e e a c h

rabbits

3 male & 3 f e m a l e e a c h

CHRONIC (2 y r s ) rats

25 male & 25 f e m a l e e a c h

dogs

3 male & 3 f e m a l e e a c h

* From A p p r a i s a l o f S a f e t y o f C h e m i c a l s i n F o o d s , D r u g s , and C o s m e t i c s . A s s o c i a t i o n o f Food and Drug O f f i c i a l s o f U.S., 1959.

10

X E N O B I O T I C S IN F O O D S A N D

TABLE I I SUBCHRONIC ORAL TOXICITY TESTING *

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch001

STANDARDIZED FACTORS* 1.

Test Duration

2.

Species

3.

Age o f Animals

4.

Number o f A n i m a l s

5.

C o n t r o l Groups

6.

Dose G r o u p s

7.

Diet

8.

Route o f A d m i n i s t r a t i o n

9.

Clinical Testing (a) (b) (c)

10.

Ophthalmological Examination Hematology Clinical

Chemistry

Observation o f Animals

11.

Gross

Necropsy

12.

Organ

Weights

13.

H i s t o p a t h o l o g i c a l E x a m i n a t i o n on a t l e a s t 32 p r i n c i p a l

*

tissues

Toxicological Principles. A d m i n i s t r a t i o n , 1982.

U.S. Food a n d Drug

FEEDS

JOSELOW

Systematic

Toxicity Testing in Foods

TABLE I I I SIGNS AND SYMPTOMS FOUND IN ANIMALS UNDERGOING TOXICOLOGIC TESTS

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch001

Signs Aggressiveness toward experimenter A l t e r e d muscle tone A l t e r a t i o n s i n cardiac rate and rhythm Catatonia (phases of stupor or excitement) Coma Convulsions to touch Paralysis Change i n p u p i l l a r y s i z e S e n s i t i v i t y to pain Skin l e s i o n s Corneal o p a c i t i e s Placing reflexes Righting r e f l e x e s Grasping r e f l e x e s Death Symptoms

Abnormal excreta Exploratory behavior Inactivity Convulsions , spontaneous Dyspnea (shortness of breath) Sedation Nystagmus (involuntary r a p i d movements of eyeballs) Cyanosis Salivation Nasal Discharge P i l o e r e c t i o n (erection of hair) Phonation (utterance of vocal sounds) Unusual p h y s i c a l p o s i t i o n s Unusual t a i l p o s i t i o n s

XENOBIOTICS IN FOODS

A N D FEEDS

TABLE IV

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch001

CLINICAL PROCEDURES COMMONLY EMPLOYED IN ANIMAL TOXICOLOGICAL TESTS

Blood chemistry studies Sodium Potassium Blood urea nitrogen Glucose Urinalysis pH and s p e c i f i c g r a v i t y Protein Glucose Ketones Crystals Blood c e l l s Bacteria Hematology Hematocrit T o t a l red blood c e l l counts T o t a l and d i f f e r e n t i a l white blood c e l l counts Organ f u n c t i o n t e s t s Bromsulfophthalein r e t e n t i o n ( l i v e r function) Thymol t u r b i d i t y ( l i v e r function) Serum a l k a l i n e phosphatase ( l i v e r function) Blood urea nitrogen (kidney function)

Figure 1. Safety decision tree. Key: + , presents socially unacceptable risk; - , does not present a socially unacceptable risk; S, metabolites known and safe; U, metabolites unknown or of doubtful safety; and ?, decision requires more evidence. (Reproduced with permission jrom Kef. 8. Copyright 1978, F o o d C o s m e t . T o x i c o l . )

Exposure

Assessment

Defined

Test Material

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch001

14

X E N O B I O T I C S IN F O O D S A N D

FEEDS

of the carcinogenic potential o f the substance. Thes a f e t y o f the s u b s t a n c e f o r humans may t h e n be e s t i m a t e d . The word " e s t i m a t e " i s u s e d a d v i s e d l y . A f t e r c o m p l e t i o n o f a l l t h e a n i m a l t e s t s , o n l y a c t u a l e x p o s u r e i n humans w i l l r e v e a l any s u b t l e o r u n f o r e s e e n t o x i c i t y . T o x i c i t y t e s t i n g c a n n o t be c o n ­ s i d e r e d c o m p l e t e u n t i l t h e s u b s t a n c e has gone t h r o u g h e x t e n s i v e u s e i n humans. A n d even t h e n , i t may n o t be c o m p l e t e , u n l e s s ade­ q u a t e r e c o r d s , f o l l o w - u p s , e t c . , have been m a i n t a i n e d and p r o s p e c ­ t i v e e p i d e m i o l o g i c a l s t u d i e s a r e made.

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch001

Literature Cited 1. Toxicants Occurring Naturally i n Foods. Report, Committee on Food Protection, National Academy of Sciences, Washington, DC, 1973. 2. Principles and Procedures f o r Evaluating the Safety of Food Additives. Food Protection Committee, Food and Nutrition Board, National Academy of Sciences, Publ. No. 750, December 1959. 3. Hazardous Substances Act. Consumer Product Safety Commission, Washington, DC, 1959. 4. Appraisal of Safety of Chemicals i n Foods, Drugs, and Cosmet­ i c s . Association of Food & Drug O f f i c i a l s of the United States, Washington, DC, 1959. 5. Toxicological Principles f o r the Safety Assessment of Direct Food Additives and Color Additives Used i n Food. U.S. Food and Drug Administration, 1982. 6. O.E.C.D. Guidelines f o r Testing of Chemicals. O.E.C.D. P u b l i ­ cations, Washington, DC, 1982. 7. Weil, C.S. and Scala, R. A. Tox. Appl. Pharmacol., 1971, 19, 276. 8. Food and Cosmetics Toxicology, 16, 9 (1978), Pergamon Press, Oxford. General Bibliography Doull, J.; Klaasen, C.; Amdur, Μ., Eds. Toxicology, The Basic Science of Poisons, Macmillan Publishing C o . , New York, 1980. Hayes, A. W. Principles and Methods of Toxicology, Raven Press, New York, 1982. Loomis, T . A. Essentials of Toxicology, 3rd ed., Lea & Febiger, New York, 1978. Mehlman, Μ. Α.; Blumenthal, H.; Shapiro, R . , Eds. Advances i n Mod­ ern Toxicology, New Concepts i n Safety Evaluation, V o l . 1, Part 1, 2. John Wiley & Son, New York, 1979. Paget, G. E., Ed. Methods i n Toxicology. F. A. Davis & Co., P h i l a ­ delphia, 1970. RECEIVED July 6, 1983

2 Protease and Amylase Inhibitors in Biological Materials JOHN R. WHITAKER

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch002

Department of Food Science and Technology, University of California, Davis, CA 95616

Proteins which specifically inhibit enzymes by forming tight inactive complexes with the enzyme are widely distributed in biological materials. With the exception of a few of the protease inhibitors and the α-amylase inhibitors, very l i t t l e work has been done on the mechanism of action of the inhibitors or of their nutritional and physiological importance. The trypsin and chymotrypsin inhibitors appear to form specific complexes with the proteases because trypsin and chymotrypsin recognize the protein inhibitors as substrates. However, the normal sequence of cataly­ tic steps cannot be completed, perhaps because of a conformational change accompanying complexation. On the other hand, the α-amylase inhibitors may not bind at the active site of α-amylase. Rather, the initial complex may undergo a conformational change which destroys the catalytic ability of the α-amylase but leaves the substrate binding ability intact. Some of the animal protease inhibitors are known to serve a protective function against proteases. There is speculation that the plant protease and amylase in­ hibitors may serve as a protection against insects and microorganisms, but this has not been proven. There is a great deal of work yet to be done on the naturally-occurring protein and peptide inhibi­ tors of enzymes. Better knowledge of their proper­ ties and their physiological, nutritional and medical roles is essential. Any compound which decreases the a c t i v i t y of an enzyme i s an i n h i b i t o r . Inhibitors of enzymes include: (a) small molecules which combine with an essential group of the active s i t e (examples include heavy metal ions, acylating and alkylating reagents) or remove an essential part of the active site (an example i s removal of essential cations by chelating agents) and compounds which sim0097-6156/83/0234-0015$09.00/0 © 1983 American Chemical Society

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch002

16

X E N O B I O T I C S IN F O O D S

AND

FEEDS

u l a t e the substrates (competitive i n h i b i t o r s , i n c l u d i n g products of the r e a c t i o n ) and (b) l a r g e polymeric molecules which i n h i b i t enzymes ( p r o t e i n and peptide i n h i b i t o r s of proteases; p r o t e i n , peptide and carbohydrate i n h i b i t o r s of α-amylases). This chapter w i l l deal only with s e l e c t e d examples from the second group. pH, temperature, denaturing agents and p r o t e o l y s i s , which decrease enzyme a c t i v i t y , are excluded from the d e f i n i t i o n of an i n h i b i t o r given above. Study of n a t u r a l l y - o c c u r r i n g enzyme i n h i b i t o r s i s of impor­ tance f o r s e v e r a l reasons. These i n c l u d e : (a) the p h y s i o l o g i c a l importance of an i n h i b i t o r i n b i o l o g i c a l m a t e r i a l , (b) the n u t r i ­ t i o n a l importance of an i n h i b i t o r when the m a t e r i a l i s consumed as a food or feed, ( c ) the use of i n h i b i t o r s to c o n t r o l enzymatic a c t i o n , such as that of polyphenol oxidase, (d) the use of i n h i b i ­ t o r s f o r a n a l y s i s and f o r p u r i f i c a t i o n purposes and (e) a b e t t e r understanding of s p e c i f i c i n t e r a c t i o n s among complex molecules such as p r o t e i n s (examples i n c l u d e antigen-antibody r e a c t i o n s , subunit i n t e r a c t i o n s i n p r o t e i n s , enzymatic a c t i o n s ) . Occurrence of Enzyme I n h i b i t o r s Compounds i n b i o l o g i c a l m a t e r i a l s which i n h i b i t proteases and amylases were noted as early as the 1930's. Kunitz and Northrop, during the p u r i f i c a t i o n of trypsinogen and t r y p s i n from beef pan­ creas, found and i s o l a t e d a t r y p s i n i n h i b i t o r from the same source ( p a n c r e a t i c secretory t r y p s i n i n h b i t o r ; 1). About the same time, Chrzaszcz and J a n i c k i (2j3) reported that there was something i n c e r t a i n plant e x t r a c t s which i n h i b i t e d α-amylase. Since that time, many enzyme i n h i b i t o r s have been discovered, p u r i f i e d and p a r t i a l l y c h a r a c t e r i z e d . Whitaker (4) has l i s t e d 54 protease i n h i b i t o r s i n animal t i s s u e s , 44 protease i n h i b i t o r s i n plant t i s s u e s , s i x protease i n h i b i t o r s i n microorganisms and some 25 i n h i b i t o r s of n o n - p r o t e o l y t i c enzymes. While these data are important i n showing the great numbers of i n h i b i t o r s present i n b i o l o g i c a l m a t e r i a l s , the numbers are rather meaningless i n most part because many i n h i b i t o r s have yet to be discovered, the numbers r e f l e c t ( i n part) i s o i n h i b i t o r s which have been reported and the i n t e r r e l a t i o n s h i p s and homology among the i n h i b i t o r s are r e l a t i v e l y unknown (see below). Discovery of Enzyme I n h i b i t o r s . Discovery of enzyme i n h i b i t o r s i n b i o l o g i c a l m a t e r i a l s occurs p r i m a r i l y i n four ways. One of the most frequent i s the observation that the percentage recovery of an enzyme a c t i v i t y during p u r i f i c a t i o n suddenly increases at one step i n the p u r i f i c a t i o n (_5). An example i s shown i n Table I. The v a l i d i t y of such an observation must be c o l l a b o r a t e d by observing a decrease i n a c t i v i t y when some of the removed f r a c t i o n i s added back to the enzyme-containing f r a c t i o n . This method only works, of course, when the a c t i v i t y of an enzyme of the b i o l o g i c a l system i s reduced by an i n h i b i t o r i n the same p r e p a r a t i o n . In the

2.

WHITAKER

Table I.

Protease

and Amylase

Inhibitors

17

E f f e c t of Presence of I n h i b i t o r on A c t i v i t y of T r y p s i n During P u r i f i c a t i o n 3

Sp.

Preparation

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch002

a

Activity

IX c r y s t a l l i z e d

100

3Χ

crystallized

120

5Χ c r y s t a l l i z e d

120

8Χ c r y s t a l l i z e d

140

3Χ c r y s t a l l i z e d ·+ ppt'd with TCA

160

K u n i t z and Northrop (1) ; c r y s t a l l i z e d from the TCA

b

b

A polypeptide i n h i b i t o r supernatant l i q u i d .

was

case of the α-amylase i n h i b i t o r s described below, they do not i n ­ h i b i t α-amylase(s) of the same source. A second i n d i c a t i o n of the presence of an i n h i b i t o r i s when experimental animals f a i l to grow as w e l l on raw as heat-treated b i o l o g i c a l m a t e r i a l s . This was e s p e c i a l l y valuable i n research on raw soybean f l o u r . A t h i r d method of d e t e c t i n g i n h i b i t o r s , of rather l i m i t e d u t i l i t y , i s the observation of m u l t i p l e p H - a c t i v i t y optima f o r what i s otherwise a pure enzyme. For example, Schwimmer (_6) ob­ served that potato i n v e r t a s e showed a double pH optima. It was discovered that the explanation of the double pH optima was due to an i n v e r t a s e i n h i b i t o r i n the potato (7). This observation i s shown schematically i n Figure 1. The f o u r t h method of d e t e c t i n g the presence of n a t u r a l l y o c c u r r i n g enzyme i n h i b i t o r s i n b i o l o g i c a l m a t e r i a l s i s to combine e x t r a c t s with a s o l u t i o n of the enzyme being t e s t e d . This i s the most systematic way. The procedure may be no more than a s e r i e s of t e s t tubes, c o n t a i n i n g the enzyme, to which b i o l o g i c a l t i s s u e e x t r a c t s are added. There are some hazards associated with t h i s method and a d d i t i o n a l experiments are r e q u i r e d . A u s e f u l technique has been the cross-wise a p p l i c a t i o n of enzyme and i n h i b i t o r to an agar p l a t e c o n t a i n i n g substrate of the enzyme. This method was o r i g i n a t e d to detect the presence of pro­ tease i n h i b i t o r s i n microorganisms ( 8 ) ; i t has since been a p p l i e d to the search f o r amylase i n h i b i t o r s ( 9 ) . The p r i n c i p l e of the technique i s shown i n Figure 2 f o r protease i n h i b i t o r s . The buf­ fered agar gel contains abut 1% c a s e i n . C e l l u l o s e paper s t r i p s saturated with the b i o l o g i c a l extract to be tested are l a i d on the gel surface i n the v e r t i c a l d i r e c t i o n . A f t e r 15-20 minutes, these s t r i p s are removed and replaced i n the h o r i z o n t a l d i r e c t i o n with c e l l u l o s e paper s t r i p s saturated with the enzyme s o l u t i o n to be t e s t e d . A f t e r 15-20 minutes, these l a t t e r s t r i p s are removed and the gel p l a t e i s incubated overnight at 25-35°C. Protease

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch002

X E N O B I O T I C S IN F O O D S A N D F E E D S

P

Figure

1. Schematic

Enzyme

Biological Extract

Figure

H

representation of effect of enzyme inhibitor curve of an enzyme.

1 ι

1 •

1

1 ι

1 ι

2

2. Schematic representation presence of protease

1 ι

1 ι

3

1 ι

1 ι

4

1 ι

1 ι

5

on

ι

pH-activity

1—r ι

6

of casein-agar plate technique for inhibitors in biological extracts.

detecting

2.

WHITAKER

Protease

and Amylase

Inhibitors

19

a c t i v i t y i s i n d i c a t e d by a white band due to h y d r o l y s i s and prec i p i t a t i o n of the c a s e i n . If the b i o l o g i c a l extract contains an i n h i b i t o r , the white band w i l l be narrow at the cross point of the two s t r i p s ( F i g u r e 2). The system can be made more s p e c i f i c by e l e c t r o p h o r e s i s of the b i o l o g i c a l extract on the c e l l u l o s e paper s t r i p p r i o r to a p p l i c a t i o n to the agar-casein g e l . Many samples can be run simultaneously by t h i s method. Types of Protease and Amylase I n h i b i t o r s

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch002

The n a t u r a l l y - o c c u r r i n g i n h i b i t o r s of proteases and amylases can be p r o t e i n s , peptides of various s i z e s and non-proteins. Protease I n h i b i t o r s . The protease i n h i b i t o r s can be enzyme s p e c i f i c and/or group s p e c i f i c . Based on t h e i r mechanism of a c t i o n , the proteases are d i v i d e d into four groups: (a) the s e r i n e proteases, (b) the s u l f h y d r y l proteases, ( c ) the carboxyl ( a c i d ) proteases, and (d) the métallo proteases. Laskowski and Kato (10) have concluded that there are no examples of i n d i v i d u a l i n h i b i t o r s which are a c t i v e against proteases from two or more of the groups. For example, human plasma ot^-trypsin i n h i b i t o r i s a c t i v e against t r y p s i n , chymotrypsin, e l a s t a s e and plasmin ( a l l s e r i n e proteases) but i t i s not a c t i v e against proteases from any of the other groups ( 1 1 ) . The t h i o l protease i n h i b i t o r of chicken egg white (papain i n h i b i t o r ; 12,13) i s a c t i v e against f i c i n , papain, bromel a i n , cathepsin B, and s t r e p t o c o c c a l protease ( a l l s u l f h y d r y l proteases), but i t i s not a c t i v e against proteases from any of the other three groups. There are examples of i n h i b i t o r s which are s p e c i f i c f o r only one protease w i t h i n a group. The best known examples i n c l u d e the Kunitz soybean ( G l y c i n e max) i n h i b i t o r (14) and i s o i n h i b i t o r s I and II of the Great Northern bean (Phaseolus v u l g a r i s ) ( 1 5 ) . Even these two examples are not c l e a r cut as there i s some small nons t o i c h i o m e t r i c combination and i n h i b i t i o n of a-chymotrypsin. Chicken (16) and Japanese q u a i l (17) egg white ovomucoids only inhibit trypsin. One p o s s i b l e exception to the above dictum that a protease i n h i b i t o r cannot i n h i b i t proteases from two or more groups i s o^macroglobulin. T h i s very l a r g e g l y c o p r o t e i n (725,000 daltons) has a very broad s p e c i f i c i t y i n that i t binds to proteases from a l l four groups (18-23). The p o s s i b i l i t y that t h i s i s due to nons p e c i f i c adsorption i s i n d i c a t e d by the observation that the enzyme-inhibitor complex r e t a i n s a c t i v i t y on small s u b s t r a t e s , although a c t i v i t y on p r o t e i n s i s l o s t (due to s t e r i c hindrance?). Other apparent exceptions are the small peptide protease i n h i b i t o r s produced by s e v e r a l species of Streptomyces. For example, the leupeptins are a c t i v e against plasmin and t r y p s i n ( s e r i n e proteases) and papain and cathepsin Β ( s u l f h y d r y l proteases)(_24). A n t i p a i n (25) and the chymostatins are a l s o a c t i v e against enzymes from both the s e r i n e and s u l f h y d r y l groups of proteases ( 2 6 ) .

XENOBIOTICS IN FOODS A N D F E E D S

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch002

20

Amylase I n h i b i t o r s . There are four types of amylases. These a r e : (a) α-amylases, (b) β-amylases, ( c ) glucoamylases and the (d) p u l lulanase-type (debranching) amylases. The only w e l l - d e s c r i b e d type of n a t u r a l l y - o c c u r r i n g p r o t e i n amylase i n h i b i t o r s are those against the α-amylases from higher animals and i n s e c t s . These i n h i b i t o r s , with the p o s s i b l e exception of the one from corn ( 2 7 ) , are not e f f e c t i v e against higher plant and m i c r o b i a l α-amylases or against the other three types of amylases. Quite r e c e n t l y , there have been d i s c u s s i o n s of the p o s s i b i l i t y of a glycoamylase-type inhibitor (28). There are a l s o small peptide i n h i b i t o r s of α-amylase found i n c e r t a i n Streptomyces ( 2 9 ) . Two carbohydrate α-amylase i n h i b i t o r s , Acarbose and Amylostatin, have been described (30-32). Their s t r u c t u r e s are shown i n Figure 3. The i n h i b i t o r s are very s i m i l a r in structure. Heterogeneity

of Protease and Amylase

Inhibitors

There are a number of well-documented examples of hetero­ geneity among the protease and amylase i n h i b i t o r s . This hetero­ geneity i s a r e s u l t of (a) i n h i b i t o r s against m u l t i p l e enzymes from a s i n g l e b i o l o g i c a l f l u i d , (b) i s o l a t i o n from d i f f e r e n t s t r a i n s ( v a r i e t i e s ) of a s p e c i e s , ( c ) i s o i n h i b i t o r s , (d) proteo­ l y s i s , ( e ) chemical m o d i f i c a t i o n d u r i n g i s o l a t i o n , and ( f ) molecu­ l a r heterogeneity. I n h i b i t o r s of D i f f e r e n t Enzymes i n a S i n g l e B i o l o g i c a l F l u i d . A few examples w i l l i l l u s t r a t e the m u l t i p l i c i t y of i n h i b i t o r s found i n b i o l o g i c a l f l u i d s (Table I I ) . Human plasma contains at l e a s t eight d i f f e r e n t types of i n h i b i t o r s , chicken egg white contains three d i f f e r e n t types, the soybean contains four d i f f e r e n t types, and the white potato s i x d i f f e r e n t types. These are c l e a r l y d i f f e r e n t compounds as they i n h i b i t q u i t e d i f f e r e n t enzymes and they can be separated from each other. I s o l a t i o n from D i f f e r e n t S t r a i n s ( V a r i e t i e s ) of a Species. Very s i m i l a r protease i n h i b i t o r s have been i s o l a t e d from s e v e r a l d i f f e r e n t s t r a i n s ( v a r i e t i e s ) of the same species or from s i m i l a r s p e c i e s . In some cases, the i n h i b i t o r s are very s i m i l a r , i n others they are q u i t e d i f f e r e n t . Three examples w i l l i l l u s t r a t e this. Ovomucoids are probably found i n a l l species of b i r d s . They are p r o t e i n s of 28,000 daltons and c o n t a i n ~20% carbohy­ d r a t e . They are q u i t e s i m i l a r i n other p r o p e r t i e s as w e l l . How­ ever, they can be q u i t e d i f f e r e n t i n t h e i r i n h i b i t o r y p r o p e r t i e s against proteases. Chicken (16) and Japanese q u a i l (17) ovomu­ coids i n h i b i t only t r y p s i n . Tinamou ovomucoid i n h i b i t s chymotryp­ s i n and s u b t i l i s i n (66) and turkey ( 67) and penguin (68) ovomu­ c o i d s i n h i b i t t r y p s i n , chymotrypsin and s u b t l l i s i n . The reason f o r t h i s d i f f e r e n c e among the ovomucoids i s due to more than one b i n d i n g s i t e f o r proteases i n the ovomucoid molecule. As shown by

Protease

and Amylase

Inhibitors

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch002

WHITAKER

Figure

3. Structures and amylostatin

of the carbohydrate α-amylase inhibitors acarbose (30,31) (32) (m = 0 to 8, η = 1 to 8, and m + η = 1 to 8).

Soybean

Chicken egg white

(Xj-Trypsin inhibitor

Human plasma

12,700

Thiol proteinase (papain) i n h i b i t o r

Bowman-Birk

8,000

21,700

4 6,500

Ovoinhibitor

Kunitz

28,000

Ovomucoid

Cathepsins Β and Η inhibitors

cathepsin Β

T r y p s i n , chymotrypsin

Trypsin

Papain, f i c i n ,

T r y p s i n , chymotrypsin, subt i l i s i n , A. oryzae protease

Trypsin

Cathepsins Β and H

F i c i n , papain, c a t h e p s i n Β and bromelain

90,000

Thiol proteinase inhibitor

T r y p s i n , chymotrypsin, l e s s e r extent plasmin Chymotrypsin

160,000

Int er-α-1ryρ s i n inhibitor

C^ protease, plasmin, k a l l i k r e i n , others

Thrombin, other s e r i n e proteases of blood c l o t t i n g sequence

Very broad

T r y p s i n , chymotrypsin, e l a s t a s e , plasmin

Specificity

α γ-Anti chymotrypsin

104,000

62,00067,000

725,000

54,000

Molecular Weight

of Protease I n h i b i t o r s i n B i o l o g i c a l F l u i d s

C^ i n a c t i v a t o r

Antithrombinheparin c o f a c t o r

2

a -Macroglobulin

Type

Source

Table I I . Examples of M u l t i p l i c i t y

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch002

55, 56

U_, 52 - 54

12, 13

50, 51

J^6, _48, _49

k]_

45, 46

j£

41_ - 44.

37^- 40

35, 36

18 - 23

11, 33, 34

Reference

m w σ 00

Ζ U *τΐ

>

CO

α

Ο Ο

τ\

ο

DO

χ m z ο

Soybean (continued)

Potato

Elastase

Papain

inhibitor

Carboxypeptidases A and Β i n h i b i t o r

pKI-56, pKI-64

Proteinase inhibitor l i b

Proteinase inhibitor H a

Chymotrypsin inhibitor I

Components I-IV

Type

Source

Table I I . (continued)

80,000

3,100

39,000

7,000-8,000

Molecular Weight 57 58 _59 60 _61, J52 63 64 65

E l a s t a s e , a l s o t r y p s i n and chymotrypsin Trypsin Chymotrypsin Chymotrypsin, nagarse, trypsin Chymotrypsin, nagarse Kallikrein Carboxypeptidases A and Β Papain,

chymotrypsin

Reference

Specificity

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch002

OS

a ft.

Co

m

Η >

X

3

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch002

24

XENOBIOTICS

IN F O O D S A N D

FEEDS

Laskowski et a l . (69), the ovomucoids contain at l e a s t three domains each with a p o t e n t i a l binding s i t e f o r a protease. Two of these s i t e s are s p e c i f i c f o r t r y p s i n and one f o r chymotrypsin ( s u b t i l i s i n binds c o m p e t i t i v e l y with chymotrypsin). In chicken, Japanese q u a i l and tinamou ovomucoids, only one of the s i t e s i s capable of binding a protease while i n turkey and penguin ovomu­ coids two binding s i t e s are expressed and i n duck ovomucoid (67) a l l three s i t e s are expressed. This i s shown d i a g r a m a t i c a l l y i n Figure 4. The t r y p s i n i n h i b i t o r s from s e v e r a l d i f f e r e n t v a r i e t i e s of Phaseolus v u l g a r i s (common bean) are q u i t e v a r i a b l e i n amino a c i d composition (Figure 5). ASN, SER and 1/2CYS are found i n the l a r g e s t amounts i n most of the i n h i b i t o r s . MET and TRP are q u i t e low i n a l l the i n h i b i t o r s and absent i n a s u b s t a n t i a l number of them. PRO i s present i n l a r g e r amounts than i n most p r o t e i n s . PHE and TYR are i n low amounts. I s o i n h i b i t o r s (Genetic Heterogeneity). The t h i r d type of h e t e r o ­ geneity i s that of i s o i n h i b i t o r s . These i s o i n h i b i t o r s , obtained from one organ or organism, have the same s p e c i f i c i t i e s f o r b i n d ­ ing proteases. However, they d i f f e r i n one or more p r o p e r t i e s such as chromatographic and e l e c t r o p h o r e t i c behavior, heat s t a ­ b i l i t y , molecular weight, or amino a c i d composition, as w e l l as q u a n t i t a t i v e l y i n binding with proteases. S n a i l epidermis contains at l e a s t s i x t r y p s i n - k a l l i k r e i n i n ­ h i b i t o r s with molecular weights ranging from 6431 to 6591 (70-72). The soybean contains two b a s i c types of protease i n h i b i t o r s , the Kunitz i n h i b i t o r of 21,500 daltons (73) and the Bowman-Birk i n ­ h i b i t o r of 7975 daltons (74). The two are quite d i f f e r e n t pro­ t e i n s as shown i n Figure 6. The Great Northern bean (Phaseolus v u l g a r i s ) has at l e a s t three t r y p s i n i s o i n h i b i t o r s ranging i n molecular weight from 8086 to 8884 (15). There are four and p o s s i b l y s i x i s o i n h i b i t o r s of t r y p s i n i n lima bean (Phaseolus lunatus)(75). Recently, Whitaker and S g a r b i e r i (76) and S g a r b i e r i and Whitaker (77) reported there are at l e a s t four i s o i n h i b i t o r s of t r y p s i n i n B r a z i l i a n pink beans (Phaseolus v u l g a r i s L. v a r . Rosinha G2). These are separable on a DEAE-cellulose column (Figure 7) but not by a f f i n i t y chromatography (76). P r o p e r t i e s of three of the i s o i n h i b i t o r s were i n v e s t i g a t e d . They are d i f f e r e n t by d i s c g e l e l e c t r o p h o r e s i s , have s l i g h t l y d i f f e r e n t amino a c i d compositions but have i d e n t i c a l molecular weights (within e x p e r i ­ mental e r r o r ) and a l l contain one residue of mannose per mol. The molecular weights are 20,000, about twice the s i z e of most BowmanB i r k type i n h i b i t o r s i n beans· They each bind two mol of t r y p s i n and one mol of chymotrypsin per mol i n h i b i t o r . One of the l a r g e s t d i f f e r e n c e s among the three i s o i n h i b i t o r s i s i n the a f f i n i t y f o r t r y p s i n and chymotrypsin (77). As shown i n Table I I I , the d i s s o ­ c i a t i o n constants f o r t r y p s i n range from 1.8 χ 10 M^ to 8.5 χ 10~ M^while those f o r chymotrypsin range from 2.8 χ 10 M_ to 10

2.

Protease

WHITAKER

and Amylase

Inhibitors

25

Figure 4. Schematic representation of multiple binding sites for proteases, and E , on an inhibitor, I. In the case of duck ovomucoid, E is trypsin and is chymotrypsin.

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch002

B

E E

A

A

5

15

5

15

5

15

5

15

5

15

5

B

15

CO

8r

5

15

5

15

5

15

5

15

5

15

5

15

INHIBITOR N U M B E R Figure 5. Amino acid composition of several protease inhibitors from soybean ( G l y c i n e m a x ) and several species and varieties of P h a s e o l u s . The results are expressed as amino acid residues per 10,000 grams. The inhibitors are 1, Kunitz soybean trypsin inhibitor; 2, Bowman-Birk soybean trypsin inhibitor; 3-6, lima bean (Phaseolus lunatus) isoinhibitors I, II, III, and IV; 7-9, French bean (Phase o l u s c o c c i n e u s ) isoinhibitors 2, 3, and 4; 10, mung bean ( P h a s e o l u s a u r e u s Roxb.) inhibitor; 11-13, Brazilian pink bean (Phaseolus vulgaris) isoinhibitors A, B, and C; 14, kidney bean (Phaseolus vulgaris) inhibitor; 15-17, Great Northern bean (Phaseolus lunatus) isoinhibitors I, II, III, and IV; 7-9, French bean (Phasvulgaris) isoinhibitor; and 19 and 20, pinto bean (Phaseolus vulgaris) isoinhibitors I and II (97).

XENOBIOTICS

IN F O O D S A N D

FEEDS

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch002

26

Figure 6. Primary structures of the Kunitz soybean trypsin inhibitor (A) and the Bowman-Birk soybean trypsin inhibitor (B). The solid black circles on the primary structure of the Bowman-Birk soybean trypsin inhibitor represent LYSSER and LEU-SER and are the binding sites for trypsin and chymotrypsin, respectively. (Reproduced with permission from (A) Ref. 73, copyright 1973, E . J . o f B i o c h . and (B) Ref. 74, copyright 1973, J . F o o d B i o c h e m J

Protease

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch002

WHITAKER

and Amylase

Inhibitors

1.2

18

~

A

g

β ο

H»

GO c\i 0.8

>^

13.5

_l

UJ

E 9.0 I

ο ζ

< CD

oc Ο·

0.5

X

0.4 ζ

liJ

0.3 Ior ω 0.2 ·ο

4

4.5

Ο

(/)

ί

m

Ho..

< 64

96

FRACTION NO.

128

160

(4.7 ml)

Figure 7. DEAE-cellulose chromatography of the trypsin-chymotrypsin isoin­ hibitors from Brazilian pink bean (Phaseolus vulgaris var. Rosinha G2. (Repro­ duced with permission from Ref 76. Copyright 1981, J . F o o d B i o c h e m . )

28

X E N O B I O T I C S IN

Table I I I .

FOODS AND

FEEDS

1^ Values for the Binding of α-Chymotrypsin and T r y p s i n with B r a z i l i a n Pink Bean I n h i b i t o r s A, Β and C (77) *i

Inhibitors A

α-Chymotrypsin (CT) 4.4

Β

2.8

C

3.0

χ

8.5

10"

1.8

1

χ

10""

χ

1

W

T r y p s i n (T)

6.8

10"

K (CT)/K (T) 1

1

χ 10~

iU

520

χ 10"

10

160

χ 10"

10

44

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch002

7

4.4 χ 10" M. The r a t i o s of K ( C T ) / K ( T ) are: A, 520; B, 160; C, 44. These binding p r o p e r t i e s c l e a r l y i n d i c a t e that the three i s o ­ i n h i b i t o r s are d i f f e r e n t and most l i k e l y are not the r e s u l t of a r t i f a c t s ( p r o t e o l y s i s or binding with phenols) produced by the p u r i f i c a t i o n procedure. Another w e l l studied example of genetic heterogeneity i s that of the wheat α-amylase i n h i b i t o r s . There appear to be at l e a s t three d i f f e r e n t molecular weight species of i n h i b i t o r s (60,000, 24,000 and 12,000) as w e l l as d i s t i n c t species w i t h i n each of these molecular weight groups. Granum and Whitaker (78) have p u r i f i e d and c h a r a c t e r i z e d three of the α-amylase i n h i b i t o r s from Anza wheat ( T r i t i c u m aestivum var. Anza). Their chemical, p h y s i ­ c a l and i n h i b i t o r y p r o p e r t i e s were q u i t e d i f f e r e n t (Table IV). They a l s o d i f f e r e d i n l y s i n e , a r g i n i n e , h i s t i d i n e , alanine ( 1 ^ L

1

Table IV. P r o p e r t i e s of Three α-Amylase I n h i b i t o r s of Wheat ( T r i t i c u m aestivum var. Anza)(78) Inhibitors Property Molecular weight Hedrick-Smith method (79) Sedimentation e q u i l i b r i u m pi S p e c i f i c i t y on α-amylases Human s a l i v a r y Hog pancreatic Bacillus subtilis A s p e r g i l l u s oryzae a

R

m

3

0.19

1^ 0.28

1^

0.55

24,000 29,000

18,500 14,500

30,000

5.9

5.2

4.2

+ + -

± -

+ -

= E l e c t r o p h o r e t i c m o b i l i t y r e l a t i v e to bromophenol blue.

2.

WHITAKER

Protease

and Amylase

Inhibitors

29

0 · 2 8 o n l y ) , v a l i n e ( R 0 . 2 8 only) and phenylalanine ( R 0 . 2 8 only) contents. They a l l had r e l a t i v e l y high contents of p r o l i n e and half-cystine. De Ponte et a l . ( 8 0 ) have r e c e n t l y proposed a model that might e x p l a i n the r e l a t i o n s h i p among a l l the known α-amylase i n h i b i t o r s i n wheat (see Figure 8 ) . How d i f f e r e n t the 1 2 , 0 0 0 d a l t o n subunits of the three i n h i b i t o r s are from the proposed a n c e s t r a l 1 2 , 0 0 0 d a l t o n molecule i s not known. U n f o r t u n a t e l y , the RJJJ 0 . 5 5 amylase i n h i b i t o r of MW 3 0 , 0 0 0 reported by Granum and Whitaker ( 7 8 ) does not f i t t h i s model.

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch002

m

m

Proteolytic Artifacts. The f o u r t h type of heterogeneity reported i s that produced by p r o t e o l y s i s . This must be of p a r t i c u l a r con­ cern i n the i s o l a t i o n of any p r o t e i n s i n c e the h y d r o l y s i s of one or two peptide bonds w i l l give r i s e to a number of products. Re­ c e n t l y , i t has been reported that some of the protease i n h i b i t o r s p r e v i o u s l y i s o l a t e d from winged bean are the r e s u l t of p r o t e o l y s i s (81). The evidence f o r t h i s i s q u i t e convincing, l e a d i n g to the p o s s i b i l i t y that some of the i s o i n h i b i t o r s reported i n the l i t e r a ­ ture are the r e s u l t of p r o t e o l y s i s . Whitaker and S g a r b i e r i ( 7 6 ) addressed t h i s i s s u e i n d e t a i l , p r o v i d i n g s e v e r a l data to i n d i c a t e that t h i s i s probably not the case f o r the i s o i n h i b i t o r s of B r a z i l i a n pink beans. Peptide mapping of i s o i n h i b i t o r s would be a v a l u a b l e t o o l i n t h i s respect, but i t has not been p r e v i o u s l y a p p l i e d to t h i s problem. Chemical M o d i f i c a t i o n . The f i f t h type of heterogeneity i s due to chemical m o d i f i c a t i o n , other than p r o t e o l y s i s , d u r i n g i s o l a t i o n of the i n h i b i t o r s . Beans, f o r example, c o n t a i n phenolic compounds and various amounts of polyphenol oxidase. I f polyphenol oxidase i s a c t i v e d u r i n g the i s o l a t i o n procedure there i s the r e a l p o s s i ­ b i l i t y that some of the products (benzoquinones) formed w i l l r e a c t with the e-amino group of l y s i n e residues of the i n h i b i t o r ( s ) , thereby producing e l e c t r o p h o r e t i c a l l y and chromatographically d i s ­ t i n c t components. P u s z t a i ( 8 2 ) p u r i f i e d an i n h i b i t o r from kidney beans which contained a f i r m l y bound p i n k i s h - b l u e pigment that was not removed by ammonium s u l f a t e p r e c i p i t a t i o n , gel f i l t r a t i o n or by chromatography on a DEAE-Sephadex column. For i n h i b i t o r s which are g l y c o p r o t e i n s (ovomucoids, some of the Phaseolus v u l g a r i s i n h i b i t o r s , the red kidney bean α-amylase i n h i b i t o r s ) , v a r i a b l e amounts of carbohydrate attached to the p r o t e i n w i l l produce i s o ­ inhibitors. Molecular Heterogeneity. The l a s t type of heterogeneity we s h a l l d i s c u s s i s that of molecular heterogeneity. This type of hetero­ geneity was mentioned under the s e c t i o n on i n h i b i t o r s from d i f f e r ­ ent v a r i e t i e s of a s p e c i e s . I t i s w e l l known that many of the i n ­ h i b i t o r s have more than one b i n d i n g s i t e f o r proteases, e i t h e r f o r the same protease or f o r d i f f e r e n t proteases. Examples of t h i s molecular heterogeneity are shown i n Table V. In the case of the b i r d egg white ovomucoids, Laskowski et a l . ( 6 9 ) have shown that

IN F O O D S A N D

FEEDS

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch002

XENOBIOTICS

Figure 8. Possible interrelationships among the protein α-amylase families from hexaploid wheats (80). All three inhibitors may have from a common ancestral protein of 12,000 daltons.

isoinhibitor originated

a

n.d. n.d.

+

-K

τι ο ο

ο

Η

δ

χ m z ο

LB

GB

ΒΒ

LB

GB

H

eu

™

10 Cys •Asp-Gln-Cy s-Ala- Cys-Thr

Continued on next page

20 t •Pr o-Pro-Gln-Cy s-•Arg­ Cys- -Ser-Asp-Lys-rSer-j-Asn30 ( V a l , Cys)Thr - A l a + S e r f l l e •Pro-Pro-Gln(Cys i l e , Cys ,Thr, Asx, 30 1 ! Cys- -As η-Hi s -Cy s -tA l a Cys-Thr -Lys-j-Ser-j-Ile- -Pr o-Pro-Gln-Cy s--Arg- Cys •Thr" •AspSer L^ 40 30 -Ile- Cys -Ala-Leu- SerMet+Arg-Leuf-Asn- Ser-Cy s-His-Ser-Ala-Cys50 ! I 40 Val)Arg-LeufAsx-Ser-Cys-His-Ser-Ala-Cys- Lys-Ser-Cys -Met- Cys -Thr-Arg- •Ser50 j ; 40 ILeutfArg-Leuf-Asp- Ser-Cy s-His-Ser-Ala-Cy s- Lys-Ser-Cys -Ile- •Cys--Thr-Leu- •Ser -

10 Ser-Gly-His-His-Glu-His-Ser ·-Thr-Asp-Glx- -Pro-Ser-Glx- Ser-Ser- Lys-Pro-Cys-

LB

BB

Asp-Asp-Glu- Ser-Ser- •Lys-Pro-Cys-

Amino Acid Sequence Homology Among the Bowman-Birk Soybean T r y p s i n I n h i b i t o r (BB: 89), Lima Bean T r y p s i n I n h i b i t o r IV (LB; 75) and Great Northern Bean T r y p s i n I n h i b i t o r I I (GB; 90)

BB

Table V I I I .

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch002

u>

ο

•?

ε-

ν;

δ

H > m 50

3

LB

GB

BB

LB

GB

BB

Table V I I I .

7

0

Asn Asp-Asp-Ly s-Glir J 80 LysfSer-Asx-Ser-Gly-Glx-Asx-Asx 80 tAsnfAsn Lys-Ser-Ser-His-Ser-Asp-Asp-Asp-Asn

!

60 50 Tyr4Pro|-Ala-Gln CysfPhe+Cys •Val-Asp-Ile-Thr-Asp-Phe+Cys-Tyr-fGlu-Pro-[Cysi 70 60 Met -fPr of-Gly-Ly s-j-Cy s fAr g-fCy s j-Leu-Asx-Thr-Thr-Asx-Ty r+Cy s-TyrfLys-Ser4Cys 70 60 ί CysIle-fPrc^Ma-Gln+C^

— ι Ly s f P r o-Ser-Glu

Continued

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch002

α τ\ m m α

Ζ

•Α ο ο σ >

η 05

Η

δ

x m ζ ο β

ON

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch002

2.

WHITAKER

Protease

and Amylase

37

Inhibitors

There appears to be sequence homology between the pineapple stem bromelain i n h i b i t o r s and some of the small molecular weight i n h i b i t o r s from the leguminosae (91). Human i n t e r - a - t r y p s i n i n ­ h i b i t o r contains two domains with great s i m i l a r i t y to the domains of the Kunitz-type i n h i b i t o r s (44, 92-94). The o v o i n h i b i t o r s from Japanese q u a i l and chicken egg whites contain s i x tandem domains which are homologous to the Kazal p a n c r e a t i c s e c r e t o r y i n h i b i t o r and to the ovomucoids (69)· Considerable homology e x i s t s w i t h i n the binding s i t e s of sev­ e r a l of the i n h i b i t o r s as shown i n Table VII. It has been sug­ gested that t r y p s i n i n h i b i t o r s require a peptide sequence of Lys-X or Arg-X located w i t h i n a loop of the p r o t e i n closed by a d i s u l ­ f i d e bond (95,96). Reduction of d i s u l f i d e bonds are known to be quite e f f e c t i v e i n destroying the i n h i b i t o r y a c t i v i t y (77). For example, a c t i v i t y of the three i s o i n h i b i t o r s from B r a z i l i a n pink beans against both t r y p s i n and chymotrypsin was l o s t when a spe­ c i f i c d i s u l f i d e bond, of the 18-21 d i s u l f i d e bonds present, was reduced ( 77). SER appears to be a requirement of the binding s i t e also f o r l y s i n e - t y p e i n h i b i t o r s (Table V I I ) . There i s homology among the binding s i t e s f o r chymotrypsin i n the i n h i b i t o r s from lima bean, soybean and runner bean (Table VII). The apparent e s s e n t i a l i t y of a serine residue i n the b i n d ­ ing s i t e f o r chymotrypsin i s also i n d i c a t e d . There appears to be much l e s s homology among the binding s i t e s of the arginine-type t r y p s i n i n h i b i t o r s (Table V I I ) . In terms of amino a c i d composition, there i s some homology among the Bowman-Birk type i n h i b i t o r s from the leguminosae as shown i n Figure 5. This appears to be so, despite the f a c t that there are some i n h i b i t o r s with molecular weights near 8000 (Bowman-Birk soybean i n h i b i t o r , lima bean i n h i b i t o r s I-IV, French bean i n h i b i t o r s 2, 3 and 4, mung bean i n h i b i t o r , kidney bean i n ­ h i b i t o r , and Great Northern bean i n h i b i t o r s I, II and I l l b ) , and others with molecular weights near 20,000 ( B r a z i l i a n pink bean i n h i b i t o r s A, Β and C, Navy bean i n h i b i t o r and pinto bean i n h i b i ­ tors I and II)(see 97_ f o r d e t a i l s ) . It c e r t a i n l y would be h e l p f u l to have t r y p t i c peptide maps and amino a c i d sequence ( i d e a l l y ) data on a l l of these i n h i b i t o r s . Recent immunochemical data (98) have shown that the α-amylase i n h i b i t o r s from s e v e r a l v a r i e t i e s of beans have great homology. Mechanism of Action of the

Protease and

Amylase

Inhibitors

Protease I n h i b i t o r s . There are unique and s p e c i f i c r e c o g n i t i o n s i t e s on the protease i n h i b i t o r s f o r t r y p s i n and chymotrypsin as shown i n Tables VI and VII. The best data are a v a i l a b l e f o r t r y p ­ s i n where i t i s known that e i t h e r a s p e c i f i c a r g i n i n e or a l y s i n e residue i n the binding s i t e of the i n h i b i t o r i s r e q u i r e d . Modifi­ c a t i o n of the a r g i n i n e (by g l y o x y l a t i o n ) or l y s i n e (by a l k y l a t i o n , etc.) residue or t h e i r removal (99) r e s u l t s i n complete loss of

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch002

38

XENOBIOTICS

IN F O O D S A N D

FEEDS

inhibitory activity. The b i n d i n g s i t e s f o r chymotrypsin appear to r e q u i r e l e u c i n e , t y r o s i n e or methionine. It i s probable that a s e r i n e residue attached to the l y s i n e or t y r o s i n e , l e u c i n e or methionine i s a l s o required (Table V I ) . However, t h i s does not appear to be the case f o r the a r g i n i n e - t y p e t r y p s i n i n h i b i t o r s . It appears, t h e r e f o r e , that t r y p s i n and chymotrypsin recognize and bind with the same amino a c i d residues i n the i n h i b i t o r s as with any s u b s t r a t e . However, net h y d r o l y s i s of peptide bonds does not occur at the pH optimum of the enzymes f o r reasons l a r g e l y unknown at t h i s time. S p e c i f i c h y d r o l y s i s of the peptide bond i n v o l v i n g the carboxyl group of the e s s e n t i a l amino a c i d residue does occur at low pH (around pH 4 ) . However, there i s l i t t l e current evidence to i n d i c a t e that t h i s h y d r o l y s i s i s a key step i n the i n h i b i t o r y process. Two types of data argue against i t s e s s e n t i a l i t y , (a) Complexation between i n h i b i t o r and chemically modified i n a c t i v e proteases i s o f t e n just as t i g h t as with the n a t i v e protease (12, 100, 101). (b) I n i t i a l l y , X-ray c r y s t a l l o g r a p h i c data on the complexes between t r y p s i n and p r o t e i n i n h i b i t o r s were i n t e r p r e t e d to i n d i c a t e that the complexes were probably adducts with a t e t r a h e d r a l intermediate s t a t e approaching a covalent bond (102, 103). However, b e t t e r refinements of the X-ray c r y s t a l l o g r a p h i c maps i n d i c a t e the d i s t a n c e s are too great for covalent bond formation. C-NMR studies appear to c o n c l u ­ s i v e l y r u l e out formation of a t e t r a h e d r a l or covalent i n t e r ­ mediate as a step i n the mechanism of i n h i b i t i o n (104-106). K i n e t i c data i n d i c a t e a conformational change may occur f o l ­ lowing formation of the i n i t i a j . complex ( 107). Such a conforma­ t i o n a l change could provide s t a b i l i t y to the complex through p r e ­ v e n t i n g i t s ready d i s s o c i a t i o n (k_2 « k_^) or i n preventing h y d r o l y s i s of the peptide bond i n the i n h i b i t o r as would occur f o r a r e g u l a r substrate (Eqn. 1). However, J i b s o n et a l .

Ε + I

v.

1

. k

- l

^

EI

2

. ^ -2

EI

f

(1)

k

(108) have r e c e n t l y shown that a conformational change probably does not occur i n t r y p s i n on binding with the Bowman-Birk soybean i n h i b i t o r or with the chick-pea t r y p s i n i n h i b i t o r . A complete explanation of why the n a t u r a l l y - o c c u r r i n g p r o t e i n protease i n h i b i t o r s are so e f f e c t i v e as i n h i b i t o r s i s s t i l l not available. α-Amylase I n h i b i t o r s . The p r o t e i n α-amylase i n h i b i t o r s form a very t i g h t complex with s a l i v a r y and p a n c r e a t i c α-amylases. For example, the complex between the red kidney bean p r o t e i n i n h i b i t o r and porcine p a n c r e a t i c α-amylase at 30°C and pH 6.9 was c a l c u l a t e d to be 3.5 χ 10~* M_ (109). The i n a c t i v e complex forms slowly, r e ­ q u i r i n g 60 to 120 minutes to reach complete r e a c t i o n depending on pH and i n h i b i t o r and enzyme concentrations (109). The red kidney 1

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch002

2.

WHITAKER

Protease

and Amylase

Inhibitors

39

bean p r o t e i n i n h i b i t o r does not i n h i b i t plant and m i c r o b i a l α-amy­ l a s e s ; only those from higher animals and i n s e c t s are i n h i b i t e d . It appears there i s an i n i t i a l r a p i d complex formed between the enzyme and i n h i b i t o r which i s s t i l l a c t i v e (110). Then, a much slower conformational change occurs (109,110) l e a d i n g to l o s s of a c t i v i t y . u n l i k e the p r o t e i n protease i n h i b i t o r s , complexation between the red kidney bean p r o t e i n α-amylase i n h i b i t o r and α-amylase does not appear to involve binding at the a c t i v e s i t e of the α-amylase. Evidence f o r t h i s i n c l u d e s a b i l i t y of the complex to bind maltose (a competitive i n h i b i t o r of α-amylase), s t a r c h , Sephadex and to s t i l l hydrolyze small s u b s t r a t e s . The red kidney bean α-amylase i n h i b i t o r contains 9-10% cov a l e n t l y bound carbohydrate. Removal of up to 70% of the carbo­ hydrate does not a f f e c t the a c t i v i t y of the i n h i b i t o r (110). The glyco groups, removed from the p r o t e i n , do not i n h i b i t α-amylase at 3.5 χ 10* times the c o n c e n t r a t i o n of the i n h i b i t o r (110). Chemical m o d i f i c a t i o n studies i n d i c a t e that h i s t i d i n e and t y r o s i n e residues i n the i n h i b i t o r may be important f o r i t s a c t i v i t y (110). In summary, our present knowledge of the mechanism of a c t i o n of the red kidney bean α-amylase i n h i b i t o r i n d i c a t e s that an i n i t i a l complex i s formed between i n h i b i t o r and enzyme which does not involve the a c t i v e s i t e of the enzyme (complex s t i l l f u l l y active). Subsequently, there i s a conformational change i n the complex which destroys the a b i l i t y of α-amylase to hydrolyze large substrates but does not prevent t h e i r binding to the enzyme. P h y s i o l o g i c a l and N u t r i t i o n a l Importance of the Protease and Amylase I n h i b i t o r s Protease I n h i b i t o r s . In animals, the p h y s i o l o g i c a l r o l e s of sev­ e r a l of the protease i n h i b i t o r s are w e l l known. The p a n c r e a t i c protease i n h i b i t o r s p r o t e c t the p a n c r e a t i c t i s s u e against prema­ ture a c t i v a t i o n of the p r o t e o l y t i c enzyme zymogens· The i n h i b i ­ t o r s a s s o c i a t e d with the blood c l o t t i n g system prevent the pre­ mature a c t i v a t i o n of the p r o t e o l y t i c enzyme zymogens c i r c u l a t i n g i n the blood at a l l times and also regulate between coagulation and f i b r i n o l y s i s . They may also be a p r o t e c t i o n against pancre­ a t i c proteases l i b e r a t e d i n t o the blood, as i n p a n c r e a t i t i s . The protease i n h i b i t o r s i n the r e s p i r a t o r y t r a c t probably serve as a p r o t e c t i o n against proteases l i b e r a t e d by granulocytes and macro­ phages brought i n as a r e s u l t of i r r i t a t i o n and/or diseased con­ d i t i o n s of the r e s p i r a t o r y t r a c t or through i n h a l a t i o n of micro­ organisms . The p h y s i o l o g i c a l r o l e of the protease i n h i b i t o r s ( e s p e c i a l l y the small peptide d e r i v a t i v e s ) i n microorganisms may, i n part, be to prevent the growth of other microorganisms . They may also be important i n the r e g u l a t i o n of p r o t e o l y s i s i n the c e l l (111-113). The p h y s i o l o g i c a l r o l e of the protease i n h i b i t o r s i n higher plants i s l e s s c l e a r even though they may account f o r 5-10% of the

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch002

40

XENOBIOTICS

IN F O O D S A N D

FEEDS

t o t a l p r o t e i n (114). The l e v e l of i n h i b i t o r s v a r i e s with the stage of growth, suggesting that the i n h i b i t o r s are p h y s i o l o g ­ i c a l l y important (114). The t h i o l protease i n h i b i t o r s of the pineapple f r u i t have a c t i v i t y against the major p r o t e o l y t i c en­ zymes, bromelains, present i n the f r u i t (115). They may a l s o serve as a defense against i n s e c t s and microorganisms (114,116)> I n f e s t a t i o n s of potatoes with Colorado potato b e e t l e l a r v a e lead to markedly increased l e v e l s of protease I n h i b i t o r - I i n the leaves (117). The n u t r i t i o n a l importance of the protease i n h i b i t o r s i n major foods i s reasonably c l e a r . It i s known that raw soybean f l o u r i n h i b i t s growth i n r a t s , chickens and some other monogastric animals (118) and death can r e s u l t (119). It i s a l s o known that the presence of soybean i n h i b i t o r i n the small i n t e s t i n e increases the s e c r e t i o n of a hormonal pancreozymic-like substance that markedly stimulates e x t e r n a l s e c r e t i o n by the pancreas (120). The presence of a c t i v e p r o t e o l y t i c enzyme i n h i b i t o r s i n the small i n t e s t i n e increases the production and s e c r e t i o n of p r o t e o l y t i c enzymes by the pancreas, presumably to compensate f o r t h e i r l o s s by complexation (121-123). This r e s u l t s i n h y p e r p l a s i a of some of the p a n c r e a t i c c e l l s and enlargement of the pancreas. Unambiguous i n t e r p r e t a t i o n of most of the data i n the l i t e r a ­ ture on the q u a n t i t a t i v e r o l e of the protease i n h i b i t o r s i n foods i s not p o s s i b l e . This i s because foods a l s o c o n t a i n other i n h i b i ­ tory substances such as hemagglutinins, amylase i n h i b i t o r s , e s t r o ­ gens and p h y t i c a c i d . Rackis (116) has suggested that the soybean t r y p s i n i n h i b i t o r appears to account f o r 30-50% of the growth r e ­ t a r d a t i o n seen on feeding raw f l o u r and probably most of the pan­ c r e a t i c enlargement. Other workers have suggested that a part of the growth r e t a r d a t i o n may be due to u n a v a i l a b i l i t y of c y s t i n e , due to the poor d i g e s t i b i l i t y of the protease i n h i b i t o r s (124). Most, but perhaps not a l l , of the protease i n h i b i t o r s are destroyed by cooking of the food. The long range consequences of feeding humans low concentrations of a c t i v e protease i n h i b i t o r s are not known. α-Amylase I n h i b i t o r s . P r o t e i n s i n h i b i t o r y of α-amylase are found i n many b i o l o g i c a l f l u i d s (_9). However, only the p r o t e i n i n h i b i ­ t o r s found i n legumes and i n wheat have been e x t e n s i v e l y i n v e s t i ­ gated. Recently, i t has been shown that a l l i n s e c t α-amylases t e s t e d , except one, are i n h i b i t e d by the red kidney bean α-amylase i n h i b i t o r (125). Y e t t e r et a l . (126) have suggested that the wheat α-amylase i n h i b i t o r s may be a c t i v e against attack of the wheat by i n s e c t s d u r i n g storage. With one exception (see below) the plant α-amylase i n h i b i t o r s do not have any a c t i v i t y against higher plant or m i c r o b i a l amylases tested (127). The three α-amylase i n h i b i t o r s of maize have been reported to i n h i b i t maize a-amylase(s), i n d i c a t i n g a p o s s i b l e p h y s i o l o g i c a l r o l e of these i n h i b i t o r s i n maize (27).

2.

WHITAKER

Protease

and Amylase

Inhibitors

41

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch002

The n u t r i t i o n a l s i g n i f i c a n c e of the α-amylase i n h i b i t o r s i s l a r g e l y unknown. I t i s known that low l e v e l s of i n h i b i t o r y a c t i v ­ i t y can be detected i n r e g u l a r l y cooked food products. When red kidney bean α-amylase i n h i b i t o r , f r e e of protease i n h i b i t o r s and hemagglutinins, was fed to rats i n a c a s e i n d i e t at the l e v e l s of 4.5 and 75 mg/rat/day, there was no decrease i n r a t e of growth of the r a t s i n r e l a t i o n to the c o n t r o l (128). Jaffê and Vega L e t t e (129) reported f e c a l s t a r c h from r a t s fed raw white kidney beans. Lang et a l . (130) reported a reduction of growth rate and i n creased f e c a l s t a r c h l e v e l s when r a t s were fed on a c a s e i n / s t a r c h d i e t c o n t a i n i n g p u r i f i e d wheat α-amylase i n h i b i t o r s . Bo-Linn et a l . (131) reported that α-amylase i n h i b i t o r , fed to humans as an impure preparation, had no e f f e c t on the c a l o r i c value of the starchy meal.

Literature Cited 1. Kunitz, M.; Northrop, J.H. J . Gen. Physiol. 1936, 19, 9911007. 2. Chrzaszcz, T.; Janicki, J . Biochem. Z. 1933, 260, 354-68. 3. Chrzaszcz, T.; Janicki, J . Biochem. J . 1934, 28, 296-304. 4. Whitaker, J.R. "Impact of Toxicology on Food Processing"; Ayres, J . C . ; Kirschman, J . C . , Eds.; Avi Publishing Co., Inc., Westport, 1981; pp. 57-104. 5. Willstätter, R.; Bamann, E . ; Rohdewald, M. Hoppe-Seyler's Ζ. Physiol. Chem. 1930, 188, 107-23. 6. Schwimmer, S. J . Theor. Biol. 1962, 3, 102-10. 7. Schwimmer, S.; Makower, R.U.; Rorem, E.S. Plant Physiol. 1961, 36, 313-16. 8. Sandvik, O. Ph.D. Thesis, Veterinary College of Norway, Oslo, 1962. 9. Fossum, K.; Whitaker, J.R. J. Nutr. 1974, 104, 930-6. 10. Laskowski, M., J r . ; Kato, I. Ann. Rev. Biochem. 1980, 49, 593-626. 11. Kress, L.F.; Laskowski, Μ., Sr. "Proteinase Inhibitors"; Fritz, H.; Tschesche, H.; Greene, L . J . ; Truscheit, E . , Eds.; Springer-Verlag, Berlin, New York, 1974; pp. 23-30. 12. Fossum, K.; Whitaker, J.R. Arch. Biochem. Biophys. 1968, 125, 367-75. 13. Sen, L.C.; Whitaker, J.R. Arch. Biochem. Biophys. 1973, 158, 623-32. 14. Birk, Y. Methods Enzymol. 1976, 45B, 700-7. 15. Wilson, K.A.; Laskowski, Μ., Sr. J . Biol. Chem. 1973, 248, 756-62. 16. Fredericq, E . ; Deutsch, H.F. J . Biol. Chem. 1949, 181, 499510. 17. Feeney, R.E.; Means, G.E.; Bigler, J.C. J . Biol. Chem. 1969, 244, 1957-60. 18. Schönenberger, M.; Schmidtberger, R.; Schultze, H.E. Z. Naturforsch. 1958, 13, 761-72.

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Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch002

2.

WHITAKER

Protease

and Amylase

Inhibitors

43

43. Steinbuch, M. Methods Enzymol. 1976, 45B, 760-72. 44. Dietl, T.; Dobrinski, W.; Hochstrasser, K. Hoppe-Seyler's Ζ. Physiol. Chem. 1979, 360, 1313-8. 45. Sasaki, M.; Minakata, K.; Yamamoto, H.; Niwa, M.; Kato, T.; Ito, N. Biochem. Biophys. Res. Commun. 1977, 76, 917-24. 46. Ryley, H.C. Biochem. Biophys. Res. Commun. 1979, 89, 871-8. 47. Lenney, J.F.; Tolan, J.R.; Sugai, W.J.; Lee, A.G. Eur. J. Biochem. 1979, 101, 153-61. 48. Bier, M.; Terminiello, L.; Duke, J.Α.; Gibbs, R.J.; Nord, F.F. Arch. Biochem. Biophys. 1953, 47, 465-73. 49. Deutsch, H.F.; Morton, J.I. Arch. Biochem. Biophys. 1961, 93, 654-60. 50. Tomimatsu, Y.; Clary, J . J . ; Bartulovich, J.J. Arch. Biochem. Biophys. 1966, 115, 536-44. 51. Liu, W.H.; Means, G.E.; Feeney, R.E. Biochim. Biophys. Acta 1971, 229, 176-85. 52. Wu, Y.V.; Scheraga, H.A. Biochemistry 1962, 1, 698-705. 53. Frattali, V.; Steiner, R.F. Biochemistry 1968, 7, 521-30. 54. Feeney, R.E.; Allison, R.G. "Evolutionary Biochemistry of Proteins: Homologous and Analogous Proteins from Avian Egg Whites, Blood Sera, Milk and Other Substances"; John Wiley & Sons, New York, 1969. 55. Odani, S.; Ikenaka, T. J. Biochem. (Tokyo) 1973, 74, 697715. 56. Ikenaka, T.; Odani, S.; Koide, T. "Proteinase Inhibitors"; Fritz, H.; Tschesche, H.; Greene, L.J.; Truscheit, E . , Eds.; Springer-Verlag, Berlin, New York, 1974; pp. 325-43. 57. Bieth, J.; Frechin, J.-C. "Proteinase Inhibitors"; Fritz, H.; Tschesche, H.; Greene, L.J.; Truscheit, Ε., Eds.; Springer-Verlag, Berlin, New York, 1974; pp. 291-304. 58. Hwang, D.L.R.; Davis Lin, K.-T.; Yang, W.-K.; Foard, D.E. Biochim. Biophys. Acta 1977, 495, 369-82. 59. Melville, J.C.; Ryan, C.A. J. Biol. Chem. 1972, 247, 344553. 60. Iwasaki, T.; Iguchi, I.; Kiyohara, T.; Yoshikawa, M. J. Biochem. (Tokyo) 1974, 75, 1387-90. 61. Iwasaki, T.; Kiyohara, T.; Yoshikawa, M. J. Biochem. (Tokyo) 1974, 75, 843-51. 62. Iwasaki, T.; Wada, J.; Kiyohara, T.; Yoshikawa, M. J. Biochem. (Tokyo) 1975, 78, 1267-74. 63. Hojima, Y.; Moriwaki, C.; Moriya, H. J. Biochem. (Tokyo) 1973, 73, 933-43. 64. Ryan, C.A.; Hass, G.M.; Kuhn, R.W. J. Biol. Chem. 1974, 249, 5495-9. 65. Rodis, P. Ph.D. Thesis, Purdue University, Lafayette, 1974. 66. Osuga, D.T.; Feeney, R.E. Arch. Biochem. Biophys. 1968, 124, 560-74. 67. Rhodes, M.B.; Bennett, N.; Feeney, R.E. J. Biol. Chem. 1960, 235, 1686-93.

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68. Osuga, D.T.; Bigler, J.C.; Uy, R.L.; Sjöberg, L . ; Feeney, R.E. Comp. Biochem. Physiol. 1974, 48B, 519-33. 69. Laskowski, M., Jr.; Kato, I . ; Kohr, W.J. "Versatility of Proteins"; Li, C.H., Ed.; Academic Press, New York, 1978; pp. 307-18. 70. Dietl, T.; Tschesche, H. Eur. J . Biochem. 1975, 58, 453-60. 71. Tschesche, H.; Dietl, T. Eur. J . Biochem. 1975, 58, 439-51. 72. Tschesche, H.; Dietl, T. Methods Enzymol. 1976, 45B, 772-85. 73. Koide, T.; Ikenaka, T. Eur. J . Biochem. 1973, 32, 417-31. 74. Odani, S.; Ikenaka, T. J . Biochem. (Tokyo) 1973, 74, 857-60. 75. Stevens, F.C.; Wuerz, S.; Krahn, J . "Proteinase Inhibitors"; Springer-Verlag, Berlin, New York, 1974; pp. 344-54. 76. Whitaker, J.R.; Sgarbieri, V.C. J . Food Biochem. 1981, 5, 197-213. 77. Sgarbieri, V.C.; Whitaker, J.R. J . Food Biochem. 1981, 5, 215-32. 78. Granum, P.E.; Whitaker, J.R. J. Food Biochem. 1977, 1, 385401. 79. Hedrick, J . L . ; Smith, A . J . Arch. Biochem. Biophys. 1968, 126, 155-64. 80. DePonte, R.; Parlamenti, R.; Petrucci, T.; Silano, V.; Tomasi, M. Cereal Chem. 1976, 53, 805-20. 81. Lorensen, E . L . ; Prevosto, R.; Wilson, K.A. Plant Physiol. 1981, 68, 88-92. 82. Pusztai, A. Biochem. J . 1966, 101, 379-84. 83. Yamamoto, M.; Ikenaka, T. J . Biochem. (Tokyo) 1967, 62, 1419. 84. Jones, G.; Moore, S.; Stein, W.H. Biochemistry 1963, 2, 6671. 85. Belitz, H.-D.; Fuchs, Α.; Nitsche, G.; Al-Sultan, T. Z. Lebensm. Unters.-Forsch. 1972, 150, 216-20. 86. Feinstein, G.; Feeney, R.E. Biochim. Biophys. Acta 1967, 140, 55-61. 87. Haynes, R.; Osuga, D.T.; Feeney, R.E. Biochemistry 1967, 6, 541-7. 88. Hory, H.-D.; Weder, J.K.P. Z. Lebensm. Unters.-Forsch. 1976, 162, 349-56. 89. Odani, S.; Ikenaka, T. J . Biochem. (Tokyo) 1972, 71, 839-48. 90. Wilson, K.A.; Laskowski, M., Sr. "Proteinase Inhibitors"; Fritz, H.; Tschesche, H.; Greene, L.J.; Truscheit, Ε., Eds.; Springer-Verlag, Berlin, New York, 1974; pp. 286-90. 91. Szilagyi, S.; Szilagyi, E. Acta Biochim. Biophys. 1978, 13, 165-70. 92. Hochstrasser, K.; Wachter, E. Hoppe-Seyler's Ζ. Physiol. Chem. 1979, 360, 1285-96. 93. Wachter, E . ; Hochstrasser, K.; Bretzel, G.; Heindl, S. Hoppe-Seyler's Z. Physiol. Chem. 1979, 360, 1297-303. 94. Wachter, E.; Hochstrasser, K. Hoppe-Seyler's Ζ. Physiol. Chem. 1979, 360, 1305-11. 95. Ozawa, K.; Laskowski, M., Jr. J . Biol. Chem. 1966, 241, 3955-61.

2.

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Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch002

102. 103. 104. 105. 106. 107. 108. 109. 110. 111. 112. 113. 114. 115. 116. 117. 118. 119. 120. 121. 122. 123. 124.

WHITAKER

Protease

and Amylase

Inhibitors

45

Laskowski, Μ., Jr.; Sealock, R.W. The Enzymes 1971, 3, 375473. Sgarbieri, V.C.; Whitaker, J.R. Adv. Food Res. 1982, 28, 93166. Pick, K.-H.; Wöber, G. Hoppe-Seyler's Ζ. Physiol. Chem. 1978, 359, 1379-84. Finkenstadt, W.R.; Laskowski, M., Jr. J. Biol. Chem. 1965, 240, PC962-3. Feinstein, G.; Feeney, R.E. J. Biol. Chem. 1966, 241, 51839. Ako, H.; Foster, R.J.; Ryan, C.A. Biochemistry 1974, 13, 132-9. Sweet, R.M.; Wright, H.T.; Janin, J.; Chothia, C.H.; Blow, D.M. Biochemistry 1974, 13, 4212-28. Tschesche, H. Angew. Chem., Int. Ed. Engl. 1973, 12, 510-1. Hunkapiller, M.W.; Forgac, M.D.; Yu, E.H.; Richards, J.H. Biochem. Biophys. Res. Commun. 1979, 87, 25-31. Baillargeon, M.W.; Laskowski, Μ., Jr.; Neves, D.E.; Porubcan, M.A.; Santini, R.E.; Markley, J.L. Biochemistry 1980, 19, 5703-10. Richarz, R.; Tschesche, H.; Wüthrich, Κ. Biochemistry 1980, 19, 5711-5. Haynes, R.; Feeney, R.E. Biochemistry 1968, 7, 2879-85. Jibson, M.D.; Birk, Y.; Bewley, T.A. Int. J. Pept. Protein Res. 1981, 18, 26-32. Powers, J.R.; Whitaker, J.R. J. Food Biochem. 1977, 1, 23960. Wilcox, E.; Whitaker, J.R., unpublished data. Saheki, T.; Matsuda, Y.; Holzer, H. Eur. J. Biochem. 1974, 47, 325-32. Lenney, J.F. J. Bacteriol. 1975, 122, 1265-73. Bünning, P.; Holzer, H. J. Biol. Chem. 1977, 252, 5316-23. Richardson, M. Phytochemistry 1977, 16, 159-69. Heinrikson, R.L.; Kézdy, F.J. Methods Enzymol. 1976, 45B, 740-51. Ryan, C.A. Ann. Rev. Plant Physiol. 1973, 24, 173-96. Green, T.R.; Ryan, C.A. Science 1972, 175, 776-7. Rackis, J.J. J. Am. Oil Chemists' Soc. 1974, 51, 161A-74A. Antunes, P.L.; Sgarbieri, V.C. J. Agric. Food Chem. 1980, 28, 935-8. Khayambashi, H.; Lyman, R.L. Am. J. Physiol. 1969, 217, 64651. Green, G.M.; Lyman, R.L. Proc. Soc. Expt'l. Biol. Med. 1972, 140, 6-12. Lyman, R.L.; Olds, B.A.; Green, G.M. J. Nutr. 1974, 104, 105-10. Schneeman, B.O.; Lyman, R.L. Proc. Soc. Expt'l. Biol. Med. 1975, 148, 897-903. Kakade, M.L.; Arnold, R.L.; Liener, I.E.; Waibel, P.E. J. Nutr. 1969, 99, 34-42.

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46

125. 126. 127. 128. 129. 130.

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131.

Powers, J.R.; Culbertson, J.D. J. Food Prot. 1982, 45, 6557. Yetter, M.A.; Saunders, R.M.; Boles, H.P. Cereal Chem. 1979, 56, 243-4. Powers, J.R.; Whitaker, J.R. J. Food Biochem. 1977, 1, 21738. Savaiano, D.A.; Powers, J.R.; Costello, M.J.; Whitaker, J.R.; Clifford, A.J. Nutr. Reports Int. 1977, 15, 443-9. Jaffé, W.G.; Vega Lette, C.L. J. Nutr. 1968, 94, 203-10. Lang, J.Α.; Chang-Hum, L.E.; Reyes, P.S.; Briggs, G.M. Fed. Proc. 1974; 33, 718. Bo-Linn, G.W.; Santa Ana, C.A.; Morawski, S.G.; Fordtran, J.S. N. Engl. J. Med. 1982, 307, 1413-6.

RECEIVED June 17, 1983

3 Antibiotics in Foods BEVERLY A. FRIEND and KHEM M. SHAHANI

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch003

Department of Food Science and Technology, University of Nebraska, Lincoln, NE 68583-0919 Antibiotic residues may occur in foods in several ways. Antibiotics can be added either directly to foods to retard spoilage and extend shelf life, or can be added indirectly through contamination from the immediate environment, through animals treated with antibiotics for medical and prophylactic purposes or animals given antibiotic treated feeds for growth. Conversely, during fermentation, certain lactic cultures synthesize "natural" antibiotics which may remain in the food. These compounds include Nisin produced by Streptococcus lactis, Diplococcin from S. cremoris, "Antibacterials" from S. thermophilus, S. diacetylactis and Leuconostoc cremoris, Bulgarican from Lactobacillus bulgaricus, Lactobrevin from L. brevis, Lactolin from L. plantarum and Acidophilin, Lactocidin and Acidolin from L. acidophilus. The increase in the incidence of antibiotic residues in the food supply poses certain public health risks including the emergence of antibiotic resistant microorganisms and possible sensitivity reactions in certain individuals. The presence of antibiotics in dairy products causes technical problems because of starter culture inhibition. On the other hand, the natural antibiotics present in fermented food products may be considered beneficial since they can increase the shelf life, and possibly inhibit the growth and toxin production of pathogenic organisms and afford protection against disease organisms to the consumer. Antibiotics are a chemically diverse group of drugs whose therapeutic properties can be related to structural and metabolic differences between microbial and mammalian cells. Because of their potent antimicrobial activity, simplicity of use and relatively low cost, antibiotics have been used widely for 0097-6156/83/0234^0047$06i)0/0 © 1983 mmsm:£miM Society Library 1155 16th St. N. w. Washington, D. C. 20036

XENOBIOTICS

48

IN F O O D S A N D F E E D S

disease c o n t r o l , food and feed p r e s e r v a t i o n and growth promot i o n , r e s u l t i n g i n s i g n i f i c a n t economic b e n e f i t s to the producer and consumer.

Table I.

I.

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch003

II.

III.

O r i g i n of A n t i b i o t i c s i n Foods

D i r e c t a d d i t i o n to foods T e c h n o l o g i c a l reasons I n d i r e c t a d d i t i o n to foods Contamination from environment Medical and p r o p h y l a c t i c treatment of animals N a t u r a l c o n s t i t u e n t of feeds Feed a d d i t i v e s f o r growth promotion or other purpose U n i n t e n t i o n a l feed a d d i t i v e s N a t u r a l c o n s t i t u e n t s of food Synthesis by l a c t i c c u l t u r e s

Source:

WHO/FAO Expert Committee on Food A d d i t i v e s ( 1 ) .

As shown i n Table I, a n t i b i o t i c s may be added to foods d i r e c t l y f o r t e c h n o l o g i c a l reasons, i n d i r e c t l y through t r e a t e d animals and feeds or n a t u r a l l y through l a c t i c s t a r t e r c u l t u r e b i o s y n t h e s i s . Recent increases i n a n t i b i o t i c use f o r food prod u c t i o n as w e l l as f o r disease c o n t r o l poses c e r t a i n r i s k s . These include the emergence of p o t e n t i a l l y dangerous a n t i b i o t i c r e s i s t a n t microorganisms, the p o s s i b i l i t y of t o x i c or a l l e r g i c r e a c t i o n s i n s e n s i t i v e i n d i v i d u a l s and/or t e c h n o l o g i c a l problems of s t a r t e r c u l t u r e i n h i b i t i o n a s s o c i a t e d with a n t i b i o t i c residues i n food products. T h i s paper summarizes the r i s k s , as w e l l as some of the b e n e f i t s , of a n t i b i o t i c s i n foods and feeds. Risks of A n t i b i o t i c s i n Foods and

Feeds

On the average, 40% of the a n t i b i o t i c s s o l d i n the United States, or more than 1.0 m i l l i o n kilograms, have been used as animal feed a d d i t i v e s ( 2 ) . According to Aschbacher (30, the f o l l o w i n g l e v e l s of a n t i b i o t i c supplementation are recommended f o r growth promotion: B a c i t r a c i n at 10-50 g/ton feed, Bambermycins at 1-2 mg/ton, Carbadox at 10-25 mg/ton, C h l o r o t e t r a c y c l i n e at 10-50 mg/ton, Erythromycin at 4-70 g/ton, Lincomycin at 1-11 g/ton, O x y t e t r a c y c l i n e at 5-50 g/ton, P e n i c i l l i n at 2-50 g/ton, T y l o s i n at 4-100 g/ton and V i r g i n i a m y c i n at 10 g/ton. Higher l e v e l s have been recommended f o r p r o p h y l a c t i c purposes. As medical and non-medical use of a n t i b i o t i c s increases, the r i s k s a s s o c i a t e d with a n t i b i o t i c s i n foods and feeds a l s o i n c r e a s e .

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch003

3.

FRIEND A N D SHAHANI

Antibiotics

49

in Foods

M i c r o b i a l r e s i s t a n c e . M i c r o b i a l r e s i s t a n c e to a n t i b i o t i c s i n feeds i s not harmful per se, but may create a p u b l i c h e a l t h hazard i f the r e s i s t a n c e i n t e r f e r e s with the c o n t r o l of a given microorganism, e s p e c i a l l y a pathogen, i n animals or humans. The Food and Drug A d m i n i s t r a t i o n (4) has declared that a n t i b i o t i c s cannot be used i n animal feeds i f : (a) a d m i n i s t r a t i o n of a n t i b i o t i c s to animals s i g n i f i c a n t l y increases the a n i mal r e s e r v o i r of pathogenic gram-negative b a c i l l i which could be t r a n s f e r r e d to humans v i a the food chain; (b) a n t i b i o t i c use s i g n i f i c a n t l y increases gram-negative b a c i l l i i n animals r e s i s t a n t to a n t i b i o t i c s used i n human medicine; or (c) i n g e s t i o n of a n t i b i o t i c residues i n foods leads to an increase of a n t i b i o t i c - r e s i s t a n t pathogenic organisms i n human f l o r a . One important c o n s i d e r a t i o n , t h e r e f o r e , i s the e f f e c t of feed a n t i b i o t i c s on the Salmonella r e s e r v o i r i n animals, since these gram-negative b a c i l l i may contaminate food products and cause i l l n e s s and death i n humans. In a d d i t i o n , a l a r g e p r o p o r t i o n of the Salmonella typhimurium organisms i s o l a t e d from humans c a r r y R f a c t o r s , the DNA-containing plasmids r e s p o n s i b l e f o r the t r a n s f e r of a n t i b i o t i c r e s i s t a n c e .

Table I I .

A n t i b i o t i c Resistance of

Salmonella i n Humans

% Resistant Serotype

s. typhimurium s. e n t e r i t i d i s s. h e i d e l b e r g s . s a i n t paul s . newport Source:

1965

1969

1974

18.5 4.2 28.6 12.5 16.7

36.7 4.9 10.5 21 20

57.6 5.8 30.0 15.9 36.3

W i n s h e l l _et__al. (9) a n d N e u ^ t _ ^ l .

(10).

The e f f e c t of a n t i b i o t i c s on the Salmonella r e s e r v o i r v a r i e s with the a n t i b i o t i c s u s c e p t i b i l i t y of the organisms. In previous studies the Salmonella r e s e r v o i r decreased when animals were i n f e c t e d with an a n t i b i o t i c - s e n s i t i v e organism (5_-7), but increased when i n f e c t e d with an a n t i b i o t i c - r e s i s t a n t organism (8)· Neu and coworkers (9-10) have confirmed that a n t i b i o t i c r e s i s t a n c e i s i n c r e a s i n g i n Salmonella i s o l a t e d from humans (Table I I ) . Resistance of S_. typhimurium to A m p i c i l l i n increased from 23.4% i n 1969 to 36.9% i n 1974, r e s i s t a n c e to Streptomycin increased from 27.3% to 45.6% and r e s i s t a n c e to T e t r a c y c l i n e increased from 12.5% to 44.8%.

50

X E N O B I O T I C S IN

Table I I I .

FOODS A N D

E f f e c t of A n t i b i o t i c Supplemented Feeds on Incidence of R e s i s t a n t E. c o l i

FEEDS

the

% Resistant

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch003

A n t i b a c t e r i a l drugs

Oxytetracycline Dihydrostreptomycin Ampicillin Neomycin Sulfamerazine Source:

Illinois Swine

89.8 93.2 52.5 20.5 82.9

Illinois Poultry

59 72 17 0 21

Illinois Beef

Montana Range Cattle*

49.1 50.0 13.2 12.3 29.2

0 0.6 1.3 0 0.6

S i e g e l et a l . (15).

Range c a t t l e , minimally control.

exposed to a n t i b i o t i c s , served

as

the

I t has a l s o been w e l l documented that a n t i b i o t i c s i n animal feeds lead to a high l e v e l of a n t i b i o t i c - r e s i s t a n t coliforms (11-15). As i l l u s t r a t e d i n Table I I I , S i e g e l et a l . (15) found that I l l i n o i s farm animals fed r a t i o n s containing a n t i b i o t i c s had more a n t i b i o t i c - r e s i s t a n t E_. c o l i than Montana range c a t t l e minimally exposed to a n t i b i o t i c s . A recent study by Hankin et a l . (16) showed that raw milk contains s u b s t a n t i a l numbers of a n t i b i o t i c - r e s i s t a n t microorganisms and that some organisms r e s i s t a n t to Streptomycin, T e t r a c y c l i n e and Polymyxin can survive p a s t e u r i z a t i o n . Several gram-negative i s o l a t e s a l s o were capable of t r a n s f e r r i n g t h e i r r e s i s t a n c e to _E. c o l i . Toxic and A l l e r g i c Reactions. While the medical use of a n t i b i o t i c s involves voluntary treatment f o r short periods of time under c o n t r o l l e d s u p e r v i s i o n , the exposure to traces of a n t i b i o t i c s i n food products i s i n v o l u n t a r y and u n c o n t r o l l e d . Although these trace l e v e l s are below the l i m i t required to cause acute t o x i c r e a c t i o n s , i t i s not known whether these t r a ces are s u f f i c i e n t to cause chronic t o x i c i t y problems (17). C e r t a i n a n t i b i o t i c s notably P e n i c i l l i n , Streptomycin, Chloramphenicol and Novobiocin are s t r o n g l y a l l e r g e n i c i n sens i t i z e d i n d i v i d u a l s . The majority of the h y p e r s e n s i t i v i t y react i o n s have occurred with P e n i c i l l i n most probably because of i t s widespread usage. S e n s i t i z a t i o n occurs most o f t e n during therap e u t i c treatment. Once an i n d i v i d u a l i s s e n s i t i z e d to P e n i c i l l i n , f o r example, as l i t t l e as 40 IU (0.024 mg) administered o r a l l y may e l i c i t a l l e r g i c r e a c t i o n s . While the only f o o d - r e l a t e d e p i -

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch003

3.

FRIEND AND

SHAHANI

Antibiotics

in Foods

51

sodes of h y p e r s e n s i t i v i t y reported i n the l i t e r a t u r e involved P e n i c i l l i n - s e n s i t i v e i n d i v i d u a l s who consumed milk containing P e n i c i l l i n (18-21), i t i s p o s s i b l e that a l l e r g i c r e a c t i o n s caused by other a n t i b i o t i c residues i n food have gone unrecognized. In order to c o n t r o l chronic t o x i c i t y and a l l e r g i c r e a c t i o n s , s a f e t y standards f o r a n t i b i o t i c residues i n food have been e s t a b l i s h e d . The WHO/FAO g u i d e l i n e s f o r a n t i b i o t i c residues i n milk, meat and egg used f o r human consumption are given i n Table IV. Chloramphenicol i s h i g h l y t o x i c and, as shown, i t s use i s forbidden f o r any purpose which might y i e l d residues i n food (1). Although not approved i n the United States, i n other c o u n t r i e s N i s i n may be added d i r e c t l y to food at 20 units/gram Q_, 64). P i m a r i c i n (Natamycin) dips to c o n t r o l mold growth on the surface of cheese r e c e i v e d recent approval from the U.S. Food and Drug A d m i n i s t r a t i o n (58). I n h i b i t i o n of S t a r t e r C u l t u r e s . The primary cause of a n t i b i o t i c residues i n milk and milk products i s the f a i l u r e of producers to withhold milk from the market f o r a s u f f i c i e n t time period f o l l o w i n g v e t e r i n a r y therapy f o r m a s t i t i s or other diseases i n dairy cattle. Consumption of antibiotic-supplemented feed may a l s o lead to residues i n the milk. These a n t i b i o t i c s are q u i t e s t a b l e and remain i n the milk even a f t e r manufacturing processses i n c l u d i n g p a s t e u r i z a t i o n , drying or f r e e z i n g . Marth and E l l i c k s o n (22), Marth (23) and Mol (17) have reviewed e x t e n s i v e l y problems i n the d a i r y i n d u s t r y a s s o c i a t e d with a n t i b i o t i c residues i n the f l u i d milk supply. The major problem has been the p a r t i a l or complete i n h i b i t i o n of a c i d production by d a i r y s t a r t e r c u l t u r e s used i n the manufacture of cheese, buttermilk, sour cream or yogurt (23)· Shahani and Harper (24) determined the minimum amount of a n t i b i o t i c needed to i n h i b i t growth of 19 stock cheese c u l t u r e s . They reported from 0.05 - 1.0 IU/ml P e n i c i l l i n and from 0.05 10.0 yg/ml Auremycin were r e q u i r e d . Whitehead and Lane (25) a l s o noted that during cheese manufacture, as l i t t l e as 0.05 IU of P e n i c i l l i n per m i l l i l i t e r of milk delayed a c i d production, while 0.5 IU/ml completely i n h i b i t e d a c i d production. Low l e v e l s of a n t i b i o t i c a l s o a f f e c t the f l a v o r and texture of the f i n a l product (17, 26), as w e l l as increase the p r o b a b i l i t y of growth of undesirable a n t i b i o t i c - r e s i s t a n t c o l i f o r m s (17, 27). Several workers (28-29) reported an increase i n the methylene blue r e d u c t i o n time when 0 . 0 5 - 0 . 5 IU P e n i c i l l i n were present per m i l l i l i t e r of milk. S i m i l a r l y , Manokidis et a l . (30) noted that P e n i c i l l i n and O x y t e t r a c y c l i n e were responsible f o r a f a l s e p o s i t i v e phosphatase t e s t i n pasteurized or p a r t i a l l y p a s t e u r i z e d milk while Streptomycin, Erythromycin and Neomycin i n h i b i t e d the phosphatase t e s t to some extent i n p a r t i a l l y p a s t e u r i z e d milk, but not i n raw milk. Although raw milk cont a i n i n g a n t i b i o t i c residues was never mistakenly i d e n t i f i e d as p a s t e u r i z e d , these authors suggested that as a "precaution" a n t i b i o t i c assays be run i n conjunction with the phosphatase t e s t .

52

XENOBIOTICS IN FOODS A N D F E E D S

Table IV.

Acceptable

Milk (ppm)

Antibiotic

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch003

Levels of A n t i b i o t i c s i n Food

Meat (ppm)

Egg (ppm)

1.

Penicillins

0-0.006

0-0.06

0-0.18

2.

Oligosaccharides Streptomycin Neomycin

0-0.2 0-0.15

0-1.0 0-0.5

0-0.5 0-0.2

3.

Chloramphenicol

0

0

0

4.

Tetracyclines Tetracycline Chlorotetracycline Oxytetracycline

0-0.1 0-0.02 0-0.1

0-0.5 0-0.05 0-0.25

0-0.3 0-0.05 0-0.3

Macrolides Erythromycin Tylosin

0-0.04 0

0-0.3 0-0.2

0-0.3 0

Polyenes Nystatin Pimaricin

0-1.1 0

0-7.1 0

0-4.3 0

0-0.2 0-28

0-0.5 0-16

0-0.5 0-110

0-0.15

0-0.5

0-0.1

5.

6.

2

7.

Siderochromes

8.

Polypeptides Nisin Polymyxin Bacitracin

9. 10.

3

Griseofuivin Novobiocin

Source:

WHO/FAO Report (J_) .

Acceptable

only f o r cheese surfaces at a l e v e l of 15 ppm.

No residue l e v e l has been e s t a b l i s h e d . N i s i n maybe used as a d i r e c t food a d d i t i v e at a l e v e l l e s s than 20 units/gram.

3.

FRIEND A N D SHAHANi

Antibiotics

in Foods

53

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch003

B e n e f i t s of A n t i b i o t i c s i n Foods and Feeds

Improved l i v e s t o c k production. For more than 25 years, a n t i b i o t i c supplementation of feeds has been used r o u t i n e l y to decrease production costs and u l t i m a t e l y consumer c o s t s . A f i v e year feeding study with swine given 100g of C h l o r o t e t r a c y c l i n e , 100g Sulfmethazine and 50g P e n c i l l i n per ton of feed demonstrated that a n t i b i o t i c supplementation markedly increases the average d a i l y gain and feed e f f i c i e n c y (Table V). Although growth responses have been observed to increase up to a l i m i t of 250g a n t i b i o t i c / t o n , lower l e v e l s are used to maximize b e n e f i t s and minimize c o s t s . The annual savings of U.S. pork consumers was estimated as $200 m i l l i o n i n 1982 (31). P r e v i o u s l y , Henry (32) reported an annual savings of $241 m i l l i o n i n production costs f o r b r o i l e r s , and $73 m i l l i o n f o r market turkeys when a n t i b i o t i c supplement a t i o n was used.

Table V.

Year

E f f e c t of A n t i b i o t i c s on Weight Gains of Swine

Avg. D a i l y Gain (g) Antibiotic Ctrl

1960 1961 1962 1963 1964 1965 Source :

263 222 186 191 200 250

413 395 359 336 322 331

Imp. (%)

57 78 93 76 61 62

Feed E f f i c i e n c y (g/g) Antibiotic Ctrl

2.13 2.08 2.15 2.99 2.71 2.77

2.11 1.85 1.81 2.18 2.36 2.28

Imp. (%)

1.0 11.1 15.8 27.1 12.9 17.7

Hays (2).

I n h i b i t i o n of pathogens and t o x i n production. Bacus and Brown (33) noted that s t a p h y l o c o c c a l food poisoning has been assoc i a t e d with d e f e c t i v e l y fermented sausage i n at l e a s t s i x instances since 1967. As shown i n Table VI, l a c t i c s t a r t e r c u l t u r e s used f o r fermented sausage produce a n t i m i c r o b i a l compounds which i n h i b i t both the growth of S t a p h y l o c o c c i and the production of e n t e r o t o x i n (34). When these c u l t u r e s are used i n combination with glucose or sucrose, they are a l s o e f f e c t i v e i n preventing the formation of t o x i n by C l o s t r i d i u m botulinum, even i n the absence of n i t r i t e (35-36).

X E N O B I O T I C S IN

54

Table VI.

Without l a c t i c s t a r t e r With l a c t i c s t a r t e r

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch003

FEEDS

L a c t i c Culture I n h i b i t i o n of Staphylococci i n Dry Sausage

Sausage formulation

Source:

FOODS A N D

Storage, 3 da Log CFU pH Toxin

8.84 6.78

Niskanen and Nurmi

5.9 5.6

+

Storage, 7 da Log CFU pH Toxin

8.88 7.53

5.7 5.3

+

(34)

Shahani and a s s o c i a t e s (37) studied the e f f e c t s of the a n t i f u n g a l a n t i b i o t i c Natamycin ( P i m a r i c i n ) on the growth of s e v e r a l mycotoxigenic molds (Table V I I ) . They found that at 1.0 ppm, Natamycin i n h i b i t e d m y c e l i a l growth from 16.0 to 23.6% depending on the mold t e s t e d . Ochratoxin production was i n h i ­ b i t e d 93.2%, p e n i c i l l i c a c i d 70.6% and p a t u l i n 97.8% at the same concentration.

Table V I I .

I n h i b i t o r y E f f e c t of Natamycin on Mold Growth and Toxin Production of _A. ochraceus NRRL 3174, _P. cyclopium NRRL 1888 and ,Ρ. patulum NRRL 989

Natamycin A. ochraceus (ppm) Growth Ocratoxin

P. cyclopium Growth Penicillic acid

P. patulum Growth P a t u l i n

(% I n h i b i t i o n ) 0 1.0 10.0 50.0 Source:

16.0 46.0 52.2

93.2 100.0 100.0

16.4 61.5 65.0

70.6 98.8 100.0

23.6 71.7 77.5

97.8 100.0 100.0

Shahani et a l . (37).

Lactobacillus acidophilus s i g n i f i c a n t l y inhibited Staphylococcus aureus, Salmonella typhimurium and enteropathic E_. c o l i when grown i n a s s o c i a t i v e c u l t u r e i n a milk t h i o medium (Table V I I I ) . Shahani et a l . (39) a l s o noted that jL. a c i d o p h i l u s and _L. b u l g a r i c u s i n h i b i t e d a number of food borne pathogens when tested by a n t i b i o t i c d i s c assay procedures.

3.

FRIEND A N D SHAHANI

Table V I I I .

in

55

Foods

I n h i b i t i o n of Pathogens by _L. a c i d o p h i l u s i n A s s o c i a t i v e Culture

Inhibition (%)

Treatment

Pathogen (CFU/ml)

Staph, aureus

Control _L. a c i d o p h i l u s

1.5 2.7

χ 10 χ 10

98.2

_S. typhimurium

Control L_. a c i d o p h i l u s

1.7 2.3

χ 10* χ 10

86.5

E.

Control L_. a c i d o p h i l u s

3.3 4.3

χ 10g χ 10

87.0

Test

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch003

Antibiotics

Culture

coli

Source:

G i l l i l a n d and

Speck

(38)

The production of n a t u r a l a n t i b i o t i c s by l a c t i c s t a r t e r c u l t u r e s has been w e l l documented (40)· Some of these compounds have been i s o l a t e d and i d e n t i f i e d as noted i n Table IX. Shahani and a s s o c i a t e s (39, 49, 56-57) have studied the production of A c i d o p h i l i n and B u l g a r i c a n by L. a c i d o p h i l u s DDS 1 and _L. b u l g a r i c u s DDS 14, r e s p e c t i v e l y . These a n t i b i o t i c s were i s o l a t e d from fermented milk using a combination of methanol and acetone e x t r a c t i o n coupled with s i l i c a g e l and Sephadex chromatography. D i f f e r e n t c u l t u r e s t r a i n s were found to vary g r e a t l y i n t h e i r production of a n t i b a c t e r i a l compounds and f a c ­ t o r s such as incubation medium, pH, temperature and had a pro­ nounced e f f e c t on a n t i b i o t i c production. M i l k was an e s s e n t i a l medium, since these organisms f a i l e d to produce a n t i b i o t i c s when grown on other s y n t h e t i c or semi-synthetic media. Approximately 30-60 μ g or 0.2 to 0.4 u n i t s of A c i d o p h i l i n per ml of aqueous s o l u t i o n caused a 50% i n h i b i t i o n i n v i t r o of a wide v a r i e t y of gram-positive and gram negative organisms (56-57).

Extended s h e l f l i f e . Several a n t i b i o t i c s i n c l u d i n g T e t r a ­ c y c l i n e s , P e n i c i l l i n , Streptomycin, B a c i t r a c i n , Neomycin and S u b t i l i n i n h i b i t food spoilage microorganisms (23). U n t i l 1967,

XENOBIOTICS

56

Table IX.

N a t u r a l A n t i b i o t i c s Produced by L a c t i c Cultures

Culture

Compound

Lactobacillus acidophilus

Acidolin Acidophilin Lactocidin "Antibiotic Substance" "Bacteriocin"

Reference

(41) (39) (42) (43-44) (45)

Lactobacillin (H 0 ) Lactobrevin

(46-47) (48)

Lactobacillus bulgaricus

Bulgarican

(49)

Lactobacillus

plantarum

Lactolin

(50)

Streptococcus

cremoris

Diplococcin

(51)

Lactobacillus brevis Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch003

IN FOODS A N D F E E D S

2

2

Streptococcus d i a c e t y l a c t i s " A n t i m i c r o b i a l Substance" Streptococcus

lactis

Streptococcus

thermophilus

Nisin "Antimicrobial Compounds"

(52)

(53-54)

(55)

C h l o r o t e t r a c y c l i n e was approved by the FDA f o r l i m i t e d use i n p o u l t r y and f i s h c h i l l water. The purpose was to extend the s h e l f l i f e of these raw products and i t was assumed that a l l residues were destroyed during the normal cooking process. The FDA subsequently rescinded t h e i r approval p r i m a r i l y because of the emergence of r e s i s t a n t microorganisms. U n t i l r e c e n t l y , no a n t i b i o t i c could be added d i r e c t l y to food f o r human consumption i n the U.S. Natamycin ( P i m a r i c i n ) has now been approved f o r use on the surface of cheese and cheese s l i c e s to extend s h e l f l i f e (58). Shahani and a s s o c i a t e s (59) noted that Natamycin treatment prolonged the s h e l f l i f e of Cottage cheese, Cheddar cheese and Parmesan cheese samples i n o c u l a t e d with t o x i g e n i c molds. Natamycin has a l s o been found e f f e c t i v e i n c o n t r o l l i n g surface mold i n I t a l i a n cheeses ( 6 0 ) .

3.

FRIEND A N D SHAHANI

Table X.

Antibiotic

E f f e c t of N i s i n on Low

Fat Dairy Spread Stored at 40°C

Storage M i c r o b i a l Count (CFU/g) Time (wk) HG S t a b l i l i z e r HG + N i s i n

0 1 3 5

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch003

Source:

15 47 14 35

X X X X

5 6 io

1 0

1 0

b

Goel et a l .

* F l a v o r Score: unacceptable.

3.7 11 33 60

57

s in Foods

X X X X

io io

2

10

3 3

HG

F l a v o r Score S t a b i l i z e r HG + N i s i n

3.1 2.6 2.5 2.7

3.0 2.6 2.6 2.4

(62)

1 - e x c e l l e n t , 2 - good, 3 - f a i r , 4 - poor, 5 -

The a n t i b i o t i c N i s i n i s i n h i b i t o r y against s e v e r a l gramp o s i t i v e S t r e p t o c o c c i , L a c t o b a c i l l i , C l o s t r i d i a , Staphylococci and B a c i l l i (61-62). Goel et a l . (63) noted that the a d d i t i o n of N i s i n increased the s h e l f l i f e of low f a t d a i r y spread (Table X). In 20 countries outside the United States, N i s i n i s permitted as a d i r e c t food a d d i t i v e (64), and one major a p p l i c a t i o n has been to prevent the growth and subsequent gas production by C l o s t r i d i a i n hard cheese and processed cheese products. In France, f o r example, Nisin-producing S t r e p t o c o c c i have been employed i n the manufacture of processed cheese. N i s i n has a l s o been studied as a p o s s i b l e a l t e r n a t i v e to n i t r i t e i n the p r e s e r v a t i o n of meats (65). Mather and Babel (66) observed that the a d d i t i o n of Leuconostoc cremoris to a Cottage cheese creaming mixture i n h i b i t e d c o l i f o r m s and prevented slime formation by Pseudomonas spp. T h i s procedure has been used commercially to extend the s h e l f l i f e of Cottage cheese (40). P o s s i b l e p r o t e c t i o n against disease. The n a t u r a l a n t i b i o t i c e f f e c t s of L^. a c i d o p h i l u s and L^. b u l g a r i c u s against human disease have received increased i n t e r e s t , e s p e c i a l l y i n Eastern Europe, Russia and Japan. In 1978, f o r example, a Russian conference reported the use of Koumiss, a fermented mare's milk product containing _L. a c i d o p h i l u s , _L. b u l g a r i c u s and Saccharomyces l a c t i s , i n the treatment of a number of diseases i n c l u d i n g n o n - s p e c i f i c and chronic lung disease, d i g e s t i v e t r a c t disease, myocardial i n f a r c t i o n , chronic c h o l e s o c y s t i t i s and chronic enterocolitis. Beck and Necheles (67) reported that _L. a c i d o p h i l u s was e f f e c t i v e i n treatment of d i f f e r e n t types of d i a r r h e a . A c i -

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch003

58

X E N O B I O T I C S IN

FOODS A N D

FEEDS

dophilus milk has been used to t r e a t j ^ . coli-mediated diarrhea i n i n f a n t s i n Yugoslavia (68), and Shigella-and-Salmonellamediated dysentery i n c h i l d r e n i n Poland (69-70). The P o l i s h researchers suggested that the production of a n t i b i o t i c substances by L^. a c i d o p h i l u s may i n part be responsible f o r the observed e f f e c t . Hamada et a l . (71) fed a L a c t o b a c i l l i - f e r m e n t e d milk known as Yakult to a group of 500 servicemen i n Japan and noted that none was i n f e c t e d with dysentery or became c a r r i e r s of Salmonella/ S h i g e l l a organisms during a 6 month p e r i o d . A c o n t r o l group had 55 p a t i e n t s with dysentery and 50 c a r r i e r s of dysentery organisms during the f i n a l month of the i n v e s t i g a t i o n . In c o n t r a s t , Pearce and Hamilton (72), Merson et a l . (73) and DeDios Pozo-Olano et a l . (74) among others have reported no s i g n i f i c a n t e f f e c t on diarrhea when L a c t o b a c i l l i were fed. C o n t r o l l e d c l i n i c a l studies using double b l i n d treatment protoc o l s with v i a b l e c u l t u r e s are required to assess whether disease p r o t e c t i o n can be achieved i n persons consuming c u l t u r e s or c u l t u r e - c o n t a i n i n g products. There i s considerable s t r a i n - t o - s t r a i n v a r i a t i o n among the l a c t i c s t a r t e r c u l t u r e s and any therapeutic e f f e c t s l i n k e d to the production of a n t i b i o t i c by one s p e c i f i c s t r a i n may not n e c e s s a r i l y apply to a l l other s t r a i n s of the same organism. Screening studies i n our laboratory (39) showed that although L^. a c i d o p h i l u s DDS 1 produced A c i d o p h i l i n and L. b u l g a r i c u s DDS 14 produced B u l g a r i c a n , none of the other s t r a i n s of these organisms produced s i g n i f i c a n t q u a n t i t i e s of a n t i b i o t i c . Commercial preparations containing s t a r t e r c u l t u r e s which produce a n t i b i o t i c s i n the laboratory a l s o may not contain s u f f i c i e n t numbers of v i a b l e organisms and/or a n t i b i o t i c to be of any b e n e f i t . F i n a l l y , these c u l t u r e s are quite f a s t i d i o u s i n t h e i r growth and metabolic requirements and therefore must not be grown or ingested i n the presence of incompatible foods or food i n g r e dients. In summary, a n t i b i o t i c residues i n foods pose c e r t a i n p o t e n t i a l r i s k s as w e l l as p o t e n t i a l b e n e f i t s . The emergence of p o s s i b l y dangerous a n t i b i o t i c r e s i s t a n c e organisms has led to the c o n s i d e r a t i o n that a n t i b i o t i c s commonly used i n humans or those which are cross r e s i s t a n t with important a n t i b i o t i c s used i n humans, be eliminated from the food supply by banning t h e i r use i n l i v e s t o c k production. A d d i t i o n a l research i n t h i s area and that of t o x i c and/or a l l e r g i c s e n s i t i v i t y r e a c t i o n s i s r e q u i r e d to make assessment of t h e i r p u b l i c h e a l t h r i s k s . Only then can c r i t i c a l r i s k / b e n e f i t d e c i s i o n s be made f o r a n t i biotics. Acknowledgments Published as Paper Number 7008, Journal S e r i e s , Nebraska A g r i c u l t u r a l Experiment S t a t i o n . Research supported, i n p a r t , by the N a t i o n a l Dairy C o u n c i l .

3.

FRIEND A N D SHAHANI

Antibiotic

s in Foods

59

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch003

Literature Cited 1. WHO/FAO Expert Committee on Food Additives. Report Series No. 430, Geneva, 1969, p. 5. 2. Hays, V. W. In "Nutrition and Drug Interrelations," Hathcock, J. N. and Coon, J., Eds.; Academic:New York, 1978, p.545. 3. Aschbacher, P. W. In "Nutrition and Drug Interrelations," Hathcock, J. N. and Coon, J., Eds.; Academic:New York, 1978, p. 630. 4. Gardner, S. Fed. Regis. 1973, 38, 9811-14. 5. Evangelisti, D. G.; English, A. R.; Girard, A. E.; Lynch, J . E.; Solomons, I. A. Antimicrob. Agents and Chemother. 1975, 8, 664-72. 6. Gutzmann, F.; Layton, H.; Simkins, K.; Jarolmen, H. Am. J. Vet. Res. 1976, 37, 649-55. 7. Jarolmen, H.; Sairk, R. J.; Langworth, B. F. J. Appl. Bacteriol. 1976, 40, 153-61. 8. Silver, R. P.; Mercer, H. D. In "Nutrition and Drug Interrelations," Hathcock, J. N. and Coon, J., Eds.; Academic:New York, 1978, p. 649. 9. Winshell, E. B.; Cherubin, C.; Winter, J.; Neu, H. C. 1969. In "Antimicrob. Agents and Chemother.", pp. 86-89. 10. Neu, H. C.; Cherubin, C. E.; Longo, E. D.; Flouton, B.; Winter, J. 1975. J . Infec. Dis. 132, 617-22. 11. Smith, H. W.; Crabb, W. E. Vet. Rec. 1957, 69, 24-30. 12. Smith, H. W. N. Z. Vet. J . 1967, 15, 153-66. 13. Loken, Κ. I.; Wagner, L. W.; Henke, C. L. Am. J . Vet. Res. 1971, 32, 1207-12. 14. Mercer, H. D.; Pocurull, D.; Gaines, S.; Wilson, S.; Bennett, J. V. Appl. Microbiol. 1971, 22, 700-5. 15. Siegel, D.; Huber, W. G.; Enloe, F. Antimicrob. Agents and Chemother. 1974, 6, 697-701. 16. Hankin, L.; Lacy, G. H.; Stephens, G. R.; Dillman, W. F. J. Food Protect. 1979, 42, 950-3. 17. Mol, H. "Antibiotics and Milk", A. A. Balkema:Rotterdam, 1975. 18. Vickers, H. R.; Bagratuni, L.; Alexander, S. Lancet 1958, 1, 351-2. 19. Erskine, D. Lancet 1958, 1, 431-2. 20. Zimmerman, M. C. Arch. Dermatol. (N.Y.) 1959, 79, 1. 21. Borrie, P.; Barret, J. Brit. Med. J. 1961, 11, 1267. 22. Marth, E. H.; Ellickson, Β. E. J. Milk Food Technol. 1959, 22, 266-72. 23. Marth, Ε. H. Residue Revs. 1966, 12, 65-161. 24. Shahani, Κ. M.; Harper, W. J. Milk Prod. J . 1958, 49, 15-16, 53-54. 25. Whitehead, H. R.; Lane, D. J. J. Dairy Res. 1956, 23, 355-60. 26. Hunter, G. J. E. J. Dairy Res. 1949, 16, 235-41.

60

27. 28. 29. 30. 31. 32.

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch003

33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54.

XENOBIOTICS IN F O O D S A N D F E E D S

Kastli, P. Schweiz. Arch. Tierheilk. 1948, 90, 685-95. Hunter, G. J. E. J . Dairy Res. 1949, 16, 149-51. Johns, C. K.; Desmaris, J . G. Can. J . Agr. Sci. 1953, 33, 91-7. Manokidis, K. S.; Alichanidis, E. S.; Varvoglis, A. G. J. Dairy Sci. 1971, 54, 335-8. "Feed Additives" Council for Agricultural Science and Technology, 1982, No. 82. Henry, W. R. "Proceedings of the Antibiotics Presentations to the U.S. Food and Drug Administration Task Force on the Use of Antibiotics in Feeds". Washington, D.C., 1970. Bacus, J. N.; Brown, W. L. Food Technol. 1981, 35, 74-8. Niskanen, Α.; Nurmi, E. Appl. Microbiol. 1976, 34, 11-20. Christiansen, L. N.; Tompkin, R. B.; Shaparis, A. B.; Johnston, R. W.; Kautler, D. A. J. Food Sci. 1975, 40, 488-90. Tanaka, N.; Traisman, E.; Lee, M. H.; Cassens, R. G.; Foster, Ε. M. J . Food Protection 1980, 43, 450-7. Shahani, K. M.; Bullerman, L. B.; Barnhardt, Η. M.; Hartung, T. E. Proc. 1st Intl. Cong. Bact. 1973, 2, 41. Gilliland, S. E.; Speck, M. L. J . Food Protection 1977, 40, 820-3. Shahani, Κ. M.; Vakil, J. R.; Kilara, A. Cult. Dairy Prod. J. 1977, 11(4), 14-7. Babel, F. J. J. Dairy Sci. 1977, 60, 815-21. Hamdan, I. Y.; Mikolajcik, Ε. M. J. Antibiotics 1974, 27, 631-6. Vincent, J. G.; Veomett, R. C.; Riley, R. I. J . Bacteriol. 1959, 78, 477-84. DeKlerk, H. C.; Coetzee, J . H. Nature 1959, 192, 340-1. Hosono, H.; Yastuki, K. Milchwissenschaft 1977, 31, 727-30. Barefoot, S. F.; Klaenhammer, T. R. J. Dairy Sci. 1981, 64 (Supplement 1), 51. Wheater, D. M.; Hirsch, Α.; Mattick, A. T. R. Nature 1951, 168, 659. Wheater, D. M.; Hirsch, Α.; Mattick, A. T. R. Nature 1952, 170, 623-4. Kavasnikov, Ε. I.; Sodenko, V. I. Mikrobiol. Zh. Kyviν. 1967, 29, 146; Dairy Science Abstracts 1967, 29, 3972. Reddy, G. V.; Shahani, Κ. M.; Friend, Β. Α.; Chandan, R. C. Cult. Dairy Prod. J. 1983, 18(2), 15-9. Kodama, R. J. Antibiotics 1952, 5, 72-4. Davey, G. P.; Richardson, B. C. Appl. Environ. Microbiol. 1981, 41, 84-9. Branen, A. L . , Go, H. C.; Genske, R. P. J . Food Sci. 1975, 40, 446-50. Mattick, A. T. R.; Hirsch, A. Nature 1944, 154, 551-4. Mattick, A. T. R.; Hirsch A. Lancet 1947, 253, 5-8.

3.

FRIEND A N D SHAHANI

55. 56. 57. 58. 59.

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch003

60. 61. 62. 63. 64. 65. 66. 67. 68. 69. 70. 71. 72. 73. 74.

Antibiotic

s in

Foods

61

Pulusani, S. R.; Rao, D. R.; Sunki, G. R. J . Food Sci. 1979, 44, 575-8. Shahani, Κ. M.; Vakil, J. R.; Kilara, A. Cult. Dairy Prod. J. 1977, 12(2), 8-11. Kilara, Α.; Shahani, K. M. J . Dairy Sci. 1978, 61, 1793-1800. Federal Register, June 22, 1982. Shahani, K. M.; Bullerman, L. B.; Evans, Τ. Α.; Arnold, R. G. Archives de L'Institut Pasteur de Tunis 1977, 3-4, 511-20. Neviani, E.; Enaldi, G. C.; Carini, S. Il Latte 1981, 6:1-9. Hawley, H. B. Food Manuf. 1957, 32, 370-6. Shahani, K. M. J . Dairy Sci. 1962, 45, 827-32. Goel, M. C.; Calbert, H. E.; Marth, Ε. H. J . Milk Food Technol. 1969, 32, 312-8. Lipinska, E. In "Antibiotics and Antibiosis in Agriculture", Woodbine, M., Ed.; Butterworths: Boston, 1977, 103-30. Rayman, M. K.; Aris, B.; Hurst, A. 1981. Appl. Environ. Microbiol. 1981, 375-80. Mather, D. W.; Babel, F. J. J. Dairy Sci. 1959, 42, 1917-26. Beck, C.; Necheles, H. Am. J. Gastroenterology 1961, 35, 522-30. Tomic-Karovic, K.; Fanjek, J. J. Annals Pediatrics 1962, 199, 625-34. Zychowicz, C.; Kowallzyk, S.; Cieplinska, T. Pediatria Polska 1977, 50(4), 429-35; Dairy Science Abstracts 38, 2382. Zychowicz, C.; Suranzynska, Α.; Siewierska, B.; Cieplinska, T. Pediatria Polska 1974, 49(8), 997-1003, Dairy Science Abstracts 38, 395. Hamada, K.; Waki, Y.; Kitagawa, T.; Uchida, K.; Chiba, H. et al. in "The Summary of Reports on Yakult". Yakult Honsha, Co., Ltd. Tokyo, Japan, 1971, pp. 54-6. Pearce, J. L.; Hamilton, J. R. J . Pediatrics 1974, 84, 261-2. Merson, M. H.; Morris, G. K.; Sack, D. A. et al. N. England J . Medicine 1976, 294, 1299-1305. De Dios Pozo-Oland, J.; Warram, J . H.; Gomez, R. G. et al. Gastroenterology, 1976, 74, 829-30.

RECEIVED June 28, 1983

4 Effects of Lipid Hydroperoxides on Food Components H. W. GARDNER

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch004

Northern Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, Peoria, IL 61604

Undesirable changes i n n u t r i t i o n a l quality of foods are i n i t i a t e d by the autoxidation or enzymic oxidation of unsaturated l i p i d s to lipid hydroperoxides. L i p i d hydroperoxides and their products of decomposition can react with food components, such as amino acids, proteins and certain other biochemicals. These reactions and the potential role of hydroperoxides i n causing mutagenicity are reviewed. Food fabrication requires many ingenious methods to prevent the development of rancidity, and the food industry largely has been successful i n this endeavor. However, the problem continues to receive serious attention by researchers. Obviously, a food that has become rancid through either enzymic oxidation or autoxidation w i l l diminish i n both n u t r i t i o n a l value and p a l a t a b i l i t y . Because rancid foods usually are rejected before consumption, i t has been debated that l i p i d oxidation i n foods has l i t t l e impact on health. It i s a concern that radical reactions i n food can cause alterations below the threshold of detection by the human senses. Considering the complexity of l i p i d peroxidation per se, the parameters added by numerous ingredients i n food pose a nearly insurmountable problem to the experimentalist. As a result, nearly a l l we know about specific molecular reactions between food biochemicals and l i p i d hydroperoxides has come from studies of model reactions employing simple systems. Data from the models must be extrapolated to the composite, and this approach i s not necessarily wholly inadequate. Obviously, certain biochemicals are more susceptible than others to radical attack and/or reaction with secondary products. This chapter not subject to U.S. copyright. Published 1983, American Chemical Society

64

X E N O B I O T I C S IN F O O D S A N D

FEEDS

W i t h s u f f i c i e n t k i n e t i c d a t a one c o u l d p r e d i c t t h e p r e d o m i n a t i n g r e a c t i o n s expected i n a complex m i x t u r e o f p o t e n t i a l reactants. I t i s the purpose of t h i s review t o : (a) b r i e f l y outline the various r a d i c a l reactions o c c u r r i n g during the course of autoxidation, (b) d i s c u s s the use o f model systems i n the study o f the e f f e c t s o f l i p i d a u t o x i d a t i o n on food i n g r e d i e n t s , p a r t i c u l a r l y p r o t e i n s , and (c) assess the p o t e n t i a l m u t a g e n i c i t y of a u t o x i d a t i o n products. Reactions

of

Autoxidation

An u n s a t u r a t e d f a t t y a c i d w i l l n o t o x i d i z e i n the presence of o r d i n a r y ground-state 0 unless a hydrogen i s f i r s t abstracted from t h e f a t t y a c i d by a r a d i c a l . This abstraction i n i t i a t e s the r a d i c a l c h a i n needed t o overcome t h e l a g o r i n i t i a t i o n phase r e q u i r e d before a u t o x i d a t i o n can a c c e l e r a t e . Since the C-H bond o f an a l l y l i c c a r b o n has a r e l a t i v e l y weak bond d i s s o c i a t i o n energy, the abstraction of this hydrogen is favored, a s i l l u s t r a t e d i n F i g u r e 1. The r e s u l t a n t a l l y l i c r a d i c a l ( p e n t a d i e n e r a d i c a l i n t h e e x a m p l e g i v e n i n F i g u r e 1) then combines w i t h 0 to produce a peroxy r a d i c a l . The p e r o x y radical i n turn propagates t h e same sequence by further Η-abstraction. The l i p i d h y d r o p e r o x i d e , thus formed, i s a l s o s u s c e p t i b l e t o h o m o l y t i c d i s s o c i a t i o n v i a R e a c t i o n s A and B .

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch004

2

2

ROOH + X ·

R 0 0 - + ΧΗ

(A)

X = R, R S , RO, R 0 0 , e t c .

ROOH

l ^ R O - + -OH

(B)

I t s h o u l d be noted t h a t R e a c t i o n A i s a n Η-abstraction, and thus i t i s u s u a l l y r e v e r s i b l e . In contrast, Reaction Β i s not r e a d i l y r e v e r s i b l e a f t e r t h e R 0 a n d ·0Η r a d i c a l s e s c a p e f r o m the solvent cage. The n e t r e s u l t o f b o t h R e a c t i o n s A and Β i s the formation of secondary products and the generation of additional radicals. Figure 2 outlines the progress of a hypothetical autoxidation of a l i p i d . The i n i t i a t i o n phase i s f o l l o w e d by r a p i d a c c u m u l a t i o n of r a d i c a l s t h a t promote both the formation and d e s t r u c t i o n of hydroperoxides. Finally, radical combination (termination) leads to nonradical secondary products. As d i s c u s s e d l a t e r , both secondary products and r a d i c a l r e a c t i o n s p e r se a r e i n v o l v e d i n food d e t e r i o r a t i o n . The m a i n p a t h w a y s t o s e c o n d a r y p r o d u c t s o f l i p i d o x i d a t i o n are described i n the following text, but the reader should r e f e r t o more c o m p r e h e n s i v e r e v i e w s on t h i s s u b j e c t ( 1 , 2 ) . e

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch004

GARDNER

Effects

of Lipid

Hydroperoxides

Figure

1. Autoxidation oflinolenic acid. Structures are abbreviated polyunsaturate. Η-Abstraction is signified by H.

Figure

2. Hypothetical

autoxidation

of a polyunsaturated time.

to show only

lipid as a function

of

X E N O B I O T I C S IN F O O D S A N D

66

FEEDS

Peroxy r a d i c a l s . L i p i d peroxy r a d i c a l s generated by Reaction A are important i n propagating other r a d i c a l s by H-abstraction (reverse of Reaction A). Weakly bonded hydrogens are p a r t i c u l a r l y susceptible to a b s t r a c t i o n by the peroxy r a d i c a l . The r a d i c a l g e n e r a t e d i n t h i s way c a n become o x i d i z e d v i a R e a c t i o n C.

X· + o — Χ 0 0 ·

(C)

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch004

2

B e c a u s e XH i s o f t e n a n o n l i p i d , w h i c h becomes o x i d i z e d i n t h e presence of peroxidizing l i p i d s , t h i s process is sometimes called "cooxidation." I n a d d i t i o n t o Η - a b s t r a c t i o n , o t h e r r e a c t i o n s compete f o r peroxy radicals, such as β-scission and i n t r a m o l e c u l a r rearrangement (3). β-Scission occurs by the reverse of R e a c t i o n C , b u t i t i s d i f f i c u l t t o s u r m i s e how t h i s r e a c t i o n would have an impact on food i n g r e d i e n t s . On t h e o t h e r h a n d , p e r o x y r a d i c a l r e a r r a n g e m e n t may h a v e more r e l e v a n c e t o f o o d systems. Rearrangement occurs i f a double bond i s p o s i t i o n e d β to the carbon bearing the peroxy group. This can lead to formation of c y c l i c peroxides (4, 5) a n d p r o s t a g l a n d i n - l i k e endoperoxides (6) b y t h e p a t h w a y s s h o w n i n F i g u r e 3. These compounds a r e b e l i e v e d t o be i m p o r t a n t i n t h e g e n e s i s of malondialdehyde (7), r a d i c a l p r o p a g a t i o n and formation of other secondary products. Peroxy r a d i c a l s can react by y e t other competing routes. For example, evidence f o r l i p i d peroxy radical combination through a tetraoxide h a s b e e n r e p o r t e d r e c e n t l y (8). Such tetraoxides could generate singlet oxygen and n o n r a d i c a l p r o d u c t s b y t h e R u s s e l l m e c h a n i s m (9) a s s h o w n i n R e a c t i o n D . R

R

H

R

R

R

H

R

I n t e r m o l e c u l a r a d d i t i o n o f t h e peroxy r a d i c a l t o a double bond a l s o i s p o s s i b l e b u t has n o t been documented i n d e t a i l f o r lipids. It has been presumed t h a t the p o l y m e r i z a t i o n of p o l y u n s a t u r a t e s may p r o c e e d i n t h i s m a n n e r .

Figure 3. The formation of hydroperoxy from the 13S-hydroperoxide of linolenic

cyclic peroxides and prostaglandin-like acid by peroxy radical rearrangement. abbreviated (6).

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch004

endoperoxides Structures are

3

X E N O B I O T I C S IN F O O D S A N D

68

FEEDS

Oxy r a d i c a l s . Heat and s i n g l e e l e c t r o n r e d u c t i o n by t r a n s i t i o n m e t a l i o n s a r e among t h e m o r e i m p o r t a n t ways l i p i d o x y r a d i c a l s are formed from h o m o l y t i c cleavage o f h y d r o p e r o x i d e s . The reactive o x y r a d i c a l i s known t o p a r t i c i p a t e i n several competitive radical processes. Like peroxy radicals, oxy r a d i c a l s have a p r o p e n s i t y f o r Η-abstraction ( R e a c t i o n E ) .

\ /

CH-O- + XH

(E)

^ C H - O H + X· /

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch004

1 Commonly, a s e l f - i n d u c e d Η - a b s t r a c t i o n , c a l l e d d i s p r o p o r t i o n a t i o n , i s observed (Reaction F ) , and indeed, f a t t y ketones and a l c o h o l s a r e f o u n d among s e c o n d a r y p r o d u c t s .

\

\

CH-O-

A

*

A

β-Scission hydrocarbons.

(Reaction

CH-OH +

\ C = 0

(F)

A

G) g e n e r a t e s

volatile

aldehydes

and

R-CH = 0 + R

\ 2

(G)

CH-0-, R

- C H = 0 + R-

A l t h o u g h v o l a t i l e s u s u a l l y do n o t e x c e e d 10-15% o f t h e o x i d i z e d lipid, the aldehyde portion of the v o l a t i l e s receives disproportionate attention because of i t s contribution to rancid odors. Oxy r a d i c a l s a d d t o o l e f i n s b y b o t h i n t e r m o l e c u l a r a n d i n t r a m o l e c u l a r mechanisms. Experimental evidence has i n d i c a t e d that intramolecular addition ( R e a c t i o n H) may b e much m o r e important than i t s intermolecular counterpart (10, 11).

(H)

4.

GARDNER

Effects

of Lipid

Hydroperoxides

Since 0 i s an e x c e l l e n t scavenger of r a d i c a l s , the r a d i c a l i s oxidized further v i a Reaction I.

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch004

2

expoxyallylic

T h e h y d r o p e r o x y g r o u p o f t h e s e c o m p o u n d s may u n d e r g o further homolysis i n the cascade to secondary products (11). In theory, combination of oxy r a d i c a l s is possible (Reaction J ) ; however, there i s l i t t l e d e t a i l e d evidence to support t h i s type of r e a c t i o n at present. RO- + X · Effect

>

ROX

(J)

o f L i p i d O x i d a t i o n on P r o t e i n

The interaction of peroxidizing lipids with protein r e c e n t l y has been reviewed by s e v e r a l i n v e s t i g a t o r s (12-17). T h i s review w i l l emphasize t h e m o l e c u l a r b a s i s f o r t h e changes i n p r o t e i n caused by exposure to p e r o x i d i z e d l i p i d . First, i t must be u n d e r s t o o d t h a t p r o t e i n c a n be a f f e c t e d by lipid hydroperoxides i n three general ways: (a) t h r o u g h formation of noncovalent complexes w i t h e i t h e r l i p i d hydroperoxide or its secondary products, (b) b y r a d i c a l r e a c t i o n s , and (c) through reactions w i t h n o n r a d i c a l secondary products. The f o r m a t i o n o f n o n c o v a l e n t complexes w i l l be i g n o r e d h e r e , b u t complexation i s probably important i n causing flavor entrainment, changes i n p r o t e i n p h y s i c a l p r o p e r t i e s and p r o m o t i o n o f c h e m i c a l reactions. Radical reactions of protein. Radical reactions of proteins promoted by l i p i d hydroperoxides fall into three general categories: (a) p r o t e i n - p r o t e i n o r l i p i d - p r o t e i n c r o s s l i n k i n g , (b) p r o t e i n s c i s s i o n , and (c) p r o t e i n o x i d a t i o n . As i l l u s t r a t e d b y a number o f r e c e n t r e p o r t s , p e r o x i d i z i n g l i p i d affects protein i n a variety of ways. F o r example, J a c k s e t a l . (18) o b s e r v e d t h a t r a n c i d o i l ( P . V . = 144) h a d no e f f e c t on the storage p r o t e i n of peanut. Other studies w i t h lysozyme (19, 2 0 ) , γ - g l o b u l i n s and a l b u m i n (21) demonstrated considerable damage to protein. Lysozyme exposed t o either peroxidizing l i n o l e i c acid or methyl linoleate resulted i n m a i n l y t h e f o r m a t i o n o f lysozyme dimers and t r i m e r s (19, 2 0 ) ,

70

X E N O B I O T I C S IN F O O D S A N D

FEEDS

as w e l l as d e n a t u r e d lysozyme (20). While Funes and K a r e l (19) observed v e r y l i t t l e l i p i d bound c o v a l e n t l y t o lysozyme, Nielsen (21) found t h a t p e r o x i d i z e d p h o s p h o l i p i d exposed to e i t h e r albumin or γ-globulin under N caused mainly p h o s p h o l i p i d p r o t e i n covalent bonds. However, N i e l s e n (21) a l s o d i d observe some d i m e r a n d h i g h e r o l i g o m e r s o f p r o t e i n . Several variables i n t h e s e i n v e s t i g a t i o n s may h a v e b e e n t h e c a u s e o f t h e d i f f e r i n g r e s u l t s , i l l u s t r a t i n g the complexity of the problem. Although the study of peroxidizing l i p i d - p r o t e i n i n t e r a c t i o n is necessary to determine the overall effects, studies of model systems e m p l o y i n g p e r o x i d i z e d l i p i d and i n d i v i d u a l amino acids also are necessary to understand the molecular basis of t h e damage. The r a d i c a l p r o c e s s e s t h a t a p p e a r t o be i m p o r t a n t w i t h amino a c i d s a r e Η - a b s t r a c t i o n , r a d i c a l c o m b i n a t i o n ( R e a c t i o n Κ ) , β - s c i s s i o n o f a m i n o a c i d o x y r a d i c a l s ( R e a c t i o n G) a n d p o s s i b l y radical addition (Reaction L ) .

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch004

2

X· + X - - * X - X

(K)

X

H

X· + / = \

(L)

The f i r s t p r o c e s s , Η - a b s t r a c t i o n , may i n i t i a t e a n i m p o r t a n t g e n e r i c r e a c t i o n o f amino a c i d s ( F i g . 4 ) . Radicals attributed to t h e α-carbon have been i d e n t i f i e d b y e l e c t r o n s p i n resonance (ESR) o f p e r o x i d i z e d p r o t e i n s ( 2 2 ) . Further reaction with 0 ( h y p o t h e t i c a l ) would l e a d t o amino a c i d h y d r o p e r o x i d e s . A d i f f e r e n t pathway t o amino a c i d h y d r o p e r o x i d e has been p r o p o s e d by Yong and K a r e l ( 2 3 ) , b u t t h e i r p r o p o s a l i n v o l v e s an i n d i r e c t pathway t o t h e α-carbon r a d i c a l . Homolysis of the hydroperoxy group would a f f o r d a n amino a c i d oxy r a d i c a l s u s c e p t i b l e t o β-scission v i a R e a c t i o n G. Thus, β-scission between the α - c a r b o n a n d t h e a m i n o g r o u p may e x p l a i n t h e i n c r e a s e i n a m i d e content of p r o t e i n t h a t has been p e r o x i d i z e d i n d r y systems, as w e l l as t h e c o i n c i d e n t p r o t e i n c h a i n s c i s s i o n observed (24). I n a d d i t i o n , m o i e t i e s v i c i n a l t o t h e α - c a r b o n may b e susceptible to radical attack, and the products o f these r e a c t i o n s may g i v e t h e f a l s e i m p r e s s i o n t h a t t h e y w e r e d e r i v e d from a t t a c k d i r e c t l y on the α-carbon. F o r example, t h e amino group c o u l d be o x i d i z e d by a f r e e r a d i c a l mechanism, and s u b s e q u e n t l y c o u l d c a u s e t h e l o s s o f t h e α-amino g r o u p . The c o n v e r s i o n o f p r o l i n e t o p r o l i n e n i t r o x i d e (25) c a n be c i t e d as a n example o f s u c h a n o x i d a t i o n . 2

Besides susceptible

the attack to radical

a t t h e α-carbon, t h e s i d e chains a r e damage. Undoubtedly, the varying

4.

GARDNER

Effects

of Lipid

Hydroperoxides

71

0

II R—CH—C—OH I NH

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch004

?

0

. II R—C—C—OH I NH 2

1.0

2

R—C—C—OH NH; Figure

4. Postulated

mechanism

of amino acid oxidation carbon.

by radical

attack of a-

X E N O B I O T I C S IN

72

FEEDS

s e n s i t i v i t y of side chains i s the reason for s e l e c t i v i t y in the p e r o x i d a t i o n o f c e r t a i n amino a c i d s . G e n e r a l l y , the most l a b i l e amino a c i d s are h i s t i d i n e , c y s t e i n e / c y s t i n e , m e t h i o n i n e , l y s i n e , t y r o s i n e and t r y p t o p h a n (14). The d e g r a d a t i o n o f c y s t e i n e p r o b a b l y p r o c e e d s t h r o u g h t h e t h i y l r a d i c a l by Η-abstraction from the t h i o l group. Strong s u l f u r s i g n a l s were o b s e r v e d b y ESR i n a m i x t u r e o f cysteine plus peroxidized methyl linoleate demonstrating the s u s c e p t i b i l i t y of t h i o l to r a d i c a l s (22). As shown i n F i g u r e 5 , cystine, various oxides of cysteine/cystine (26-28), alanine and H S (27) are products. G l u t a t h i o n e p e r o x i d i z e d by l i p i d hydroperoxide also afforded the disulfide and o x i d e s of glutathione (29). The absence of 0 from a r e a c t i o n of linoleic acid hydroperoxide plus cysteine caused an i n t e r e s t i n g s h i f t in products. Instead of c y s t i n e and c y s t e i n e / c y s t i n e oxides, c y s t i n e and l i p i d - c y s t e i n e adducts were i d e n t i f i e d from the reaction mixture (30). T h e RS» p l u s RS« a n d R S * p l u s R* c o m b i n a t i o n s w e r e f a v o r e d b e c a u s e 0 was n o t p r e s e n t t o s c a v e n g e b o t h t h e l i p i d r a d i c a l s (R*) and t h i y l r a d i c a l s ( F i g . 5 ) . The d e t a i l e d mechanism proposed f o r the c o m b i n a t i o n r e a c t i o n is g i v e n i n F i g u r e 6. The e p o x y a l l y l i c r a d i c a l shown a t t h e t o p of Figure 6 arises from lipid oxy radical rearrangement (Reaction H). We h a v e r e c e n t l y i s o l a t e d t h e epoxyene-cysteine adduct by u s i n g a r e a c t i o n system devoid of p r o t i c solvents (31) . In protic solvent the epoxide r e a d i l y s o l v o l y z e s by anchimeric a s s i s t a n c e of the t h i y l ether i n t o the f i n a l products shown i n t h e f i g u r e . T h i s new d a t a r e f u t e s a mechanism I proposed e a r l i e r (14). Such a r e a c t i o n p o s s i b l y c o u l d account for a l i p i d to p r o t e i n c r o s s l i n k ; however, proof of this p a r t i c u l a r l i p i d - p r o t e i n bond r e m a i n s t o be d e m o n s t r a t e d . As shown i n F i g u r e 7, t h e d e g r a d a t i o n o f t r y p t o p h a n by p e r o x i d i z i n g m e t h y l l i n o l e a t e has been r e p o r t e d by Yong e t a l . (32) . T h e y p o s t u l a t e d t h a t t h e i n i t i a t i n g e v e n t was a r a d i c a l a d d i t i o n of a h y d r o x y l (or l i p i d oxy) r a d i c a l to the indole r i n g ; h o w e v e r , o t h e r s ( 3 3 , 34) have d e m o n s t r a t e d t h a t f o r m a t i o n of a hydroperoxy g r o u p a t c a r b o n - 3 o f t h e i n d o l e r i n g was intermediate i n the o x i d a t i o n of i n d o l e d e r i v a t i v e s by v a r i o u s oxidants. T h i s l a t t e r p a t h w a y seems a more p l a u s i b l e r o u t e t o the p r o d u c t s observed by Yong et a l . (32). S c h a i c h and K a r e l (22) p o s t u l a t e d t h a t a n u n s p e c i f i e d t r y p t o p h a n r a d i c a l was a major c o n t r i b u t o r t o p r o t e i n ESR b e c a u s e t h e ESR s i g n a l o f peroxidized nonsulfhydryl protein strongly resembled the signal of peroxidized tryptophan. Their observation may indicate that a resonance-stabilized indole radical is also possible. H i s t i d i n e w i t h p e r o x i d i z i n g l i p i d was a l t e r e d b o t h a t t h e α-carbon and the i m i d a z o l e s i d e c h a i n (23, 3 5 ) . Histamine, imidazole acetic a c i d and i m i d a z o l e lactic acid evidently 2

2

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch004

FOODS A N D

2

4.

GARDNER

Effects

of Lipid

73

Hydroperoxides

RSH

[RS«]

Alanine HS Aldehyde adducts (Thiazolidines)

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch004

2

RSSR

RS0 H 2

RSR

RSO3H

RS0 SR 2

Figure

5. Pathways

of cysteine

(RSH) degradation lipid.

by exposure

to

peroxidizing

Figure 6. Mechanism of cysteine-fatty acid adduct formation from the reaction of 13-hydroperoxylinoleic acid and cysteine in the absence of 0 . The epoxyallylic radical at the top originates from the oxydiene radical of 13-hydroperoxylinoleic acid (abbreviated structure) and RS · is the cysteine thiyl radical. (Reproduced with permission from Ref. 28.) 2

74

X E N O B I O T I C S IN F O O D S A N D

m

-Si Su

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch004

-««» Ο

•S

•S

f0 "δ

1 Ci,

Ο

& s; Q

-s:

I

•I ε

FEEDS

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch004

4.

GARDNER

Effects of Lipid

75

Hydroperoxides

arose from a t t a c k on o r v i c i n a l t o the α-carbon. Degeneration o f t h e i m i d a z o l e r i n g was i n t i m a t e d b y t h e f o r m a t i o n o f v a l i n e and a s p a r t i c a c i d . Additional research with the h i s t i d i n e derivatives, h i p p u r y l h y s t i d y l l e u c i n e and N - b e n z o y l h i s t i d i n e , accentuated the degradation of the imidazole group (36). These derivatives were d e s i g n e d t o model the s t r u c t u r a l environment of h i s t i d y l residues i n p r o t e i n , thus attack on t h e s i d e c h a i n may b e m o r e i m p o r t a n t i n p r o t e i n s t h a n w i t h free histidine. Accordingly, the main products from peroxidation o f N - b e n z o y l h i s t i d i n e were N-benzoylasparagine and N - b e n z o y l a s p a r t i c a c i d . The products from degeneration of lysine caused by p e r o x i d i z i n g m e t h y l l i n o l e a t e (13) a r e shown i n F i g u r e 8 . The structures of the products are indicative of r a d i c a l reaction a t b o t h t h e α-carbon and t h e s i d e c h a i n . Of p a r t i c u l a r i n t e r e s t is 1,10-diamino-l,10-dicarboxydecane, which p o t e n t i a l l y could provide a c r o s s l i n k between l y s i n y l residues i n proteins. Presumably, a C-6 r a d i c a l of 2-aminohexanoic a c i d originated from s c i s s i o n o f l y s i n e ε-amino g r o u p , a n d s e l f - c o m b i n a t i o n o f the C-6 r a d i c a l generated the dimer. The ε-amino group a l s o provides the site f o r c r o s s l i n k i n g by malondialdehyde as explained i n the following section. F i n a l l y , m e t h i o n i n e a n d t y r o s i n e a r e known t o be s e n s i t i v e to p e r o x i d a t i o n . M e t h i o n i n e was o x i d i z e d t o m e t h i o n i n e s u l f o x i d e in the presence of peroxidizing methyl linoleate (13) or peroxidizing o i l (37), i l l u s t r a t i n g the ease of radical i n i t i a t i o n on s u l f u r s u b s t i t u e n t s . The r a d i c a l d e s t r u c t i o n o f t y r o s i n e i s k n o w n ( 3 8 ) , b u t I am n o t a w a r e o f a n y s t u d i e s t h a t specifically subject tyrosine to peroxidized lipid. E x t r a p o l a t i o n from o t h e r r a d i c a l r e a c t i o n s of t y r o s i n e i n d i c a t e s that the i n i t i a l event i s Η-abstraction of the phenol to a f f o r d a phenoxy r a d i c a l . Effect of nonradical oxidation products on p r o t e i n . The aldehydes formed from l i p i d a u t o x i d a t i o n b y R e a c t i o n G have a p r o p e n s i t y t o r e a c t w i t h amino groups t o form a S c h i f f base (Reaction M). R -CH = 0 + R-NH 1

2

•

R - C H= N-R 1

(M)

The i m p l i c a t i o n s o f S c h i f f b a s e f o r m a t i o n i n b i o l o g i c a l s y s t e m s have b e e n r e v i e w e d i n more d e t a i l e l s e w h e r e ( 1 2 , 1 4 , 1 7 ) ; t h u s , t h i s aspect o f l i p i d o x i d a t i o n w i l l be b r i e f . With p r o t e i n s , S c h i f f base formation w i l l occur o n l y w i t h amino a c i d r e s i d u e s p o s s e s s i n g a s i d e c h a i n amino group, i n c l u d i n g , o f c o u r s e , t h e amino t e r m i n u s . The ε-amino group of lysine i s important i n this regard, and t h e l o s s of bioavailability of l y s i n e by S c h i f f base formation is a n u t r i t i o n a l concern.

XENOBIOTICS IN FOODS A N D F E E D S

0

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch004

^Peroxidation

,0H

Aspartic

0 NH

?

yi^OH

γ

Figure

8. Products

of lysine exposed

0Η

Alanine

Glycine

to peroxidizing

methyl

linoleate (13).

4.

GARDNER

Effects

of Lipid

Hydroperoxides

The bifunctional malondialdehyde has c r o s s l i n k i n g as i l l u s t r a t e d by R e a c t i o n N.

caused

protein

0 = (N) R - N H - C H = C H - C H = NR Other derivatives from r e a c t i o n a c i d s have been d e s c r i b e d (39).

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch004

Radical Reactions

of

of

malondialdehyde

and

amino

Nonproteins

R a d i c a l s i n i t i a t e d b y l i p i d p e r o x i d a t i o n a f f e c t a number of nonprotein biochemicals. I n g e n e r a l , most o f t h e l a b i l e compounds a r e c h a r a c t e r i z e d as p o s s e s s i n g a n e a s i l y a b s t r a c t a b l e hydrogen. Accordingly, a n t i o x i d a n t s and Η-donors, such as α-tocopherol, a s c o r b i c a c i d and g l u t a t h i o n e , f a l l i n t o this category. L i p i d hydroperoxide caused the o x i d a t i o n of α-tocopherol to α-tocopherolquinone through an u n i d e n t i f i e d intermediate (40). P o r t e r e t a l . (41) proposed a mechanism o f o x i d a t i o n t h a t i n c l u d e s an i n t e r m e d i a t e from c o m b i n a t i o n o f α-tocopherol semiquinone w i t h a peroxy r a d i c a l (Reaction 0 ) .

(ο)

The a b s e n c e o f 0 causes a s h i f t i n products to the formation of α - t o c o p h e r o l - l i p i d adducts (42, 43) v i a t h e c o m b i n a t i o n o f a l i p i d oxy r a d i c a l w i t h α-tocopherol semiquinone (Reaction P ) . 2

X E N O B I O T I C S IN F O O D S A N D

78

FEEDS

Reaction Ρ i s very s i m i l a r to the combination reaction of l i p i d oxy r a d i c a l w i t h the cysteine t h i y l r a d i c a l ( F i g . 6 ) . Both combination r e a c t i o n s proceed o n l y i n the absence o f 0 , i m p l y i n g t h e 0 e f f e c t i v e l y competes f o r t h e r a d i c a l s i n v o l v e d . Under c e r t a i n c o n d i t i o n s a s c o r b i c a c i d i s an a n t i o x i d a n t probably because i t r e a d i l y loses Η to a b s t r a c t i o n . Attention a l s o has been given t o the p r o o x i d a t i v e e f f e c t of a s c o r b i c acid i n the presence of t r a n s i t i o n metal ions (44). It is thought that ascorbic a c i d reduces metal ions which i n t u r n a r e more e f f e c t i v e i n c a t a l y z i n g l i p i d o x i d a t i o n . Consequently, a s c o r b i c a c i d becomes o x i d i z e d t o d e h y d r o a s c o r b i c a c i d . The d e s t r u c t i o n o f β-carotene d u r i n g l i p i d peroxidation i s r e a d i l y observed by bleaching of the carotene color (44). Presumably, β-carotene o x i d a t i o n i s i n i t i a t e d by H - a b s t r a c t i o n , and such a mechanism has been proposed f o r t h e c o o x i d a t i o n o f carotenoids during the lipoxygenase catalyzed oxidation of p o l y u n s a t u r a t e d f a t t y a c i d s ( 4 5 ) , a s shown b y R e a c t i o n Q. 2

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch004

2

R00-

ROOH (Q)

carotene

polyene

Mutagenicity Induced by L i p i d

r a di i c a l

y

.

oxidized carotene

Oxidation

F r e e r a d i c a l o x i d a t i o n i n v i v o h a s b e e n much t o u t e d as a detriment to both h e a l t h and l i f e . Indeed, aberrant free r a d i c a l r e a c t i o n s have been c i t e d as c o n t r i b u t o r s t o a g i n g (46, 47) and cancer (47, 4 8 ) , b u t u n e q u i v o c a l evidence for these claims often i s l a c k i n g . The c o n n e c t i o n between free r a d i c a l s and t h e p r o m o t i o n o f cancer has r e c e i v e d t h e most attention. As d i s c u s s e d l a t e r , the evidence is compelling that lipid hydroperoxide activates c e r t a i n carcinogens by cooxidation. A d i r e c t mutagenic e f f e c t of l i p i d hydroperoxides has b e e n s o u g h t f o r some t i m e w i t h v a r y i n g s u c c e s s . Recently, the Ames t e s t h a s b e e n u t i l i z e d t o d e m o n s t r a t e w e a k m u t a g e n i c i t y of b o t h p e r o x i d i z e d f a t t y a c i d (49) and i s o l a t e d hydroperoxides

4.

GARDNER

Effects

of Lipid

Hydroperoxides

79

of methyl l i n o l e a t e (50). B e c a u s e cumene h y d r o p e r o x i d e and t - b u t y l h y d r o p e r o x i d e were a l s o f o u n d t o be m u t a g e n i c , while p e r o x i d e s , p e r a c i d s and H 0 w e r e n o t , t h e m u t a g e n i c i t y was attributed to the hydroperoxide group (50). As shown i n T a b l e I , we h a v e a l s o o b s e r v e d w e a k m u t a g e n i c i t y f o r methyl 13-hydroperoxylinoleate by the Ames test (51). A third l a b o r a t o r y has f a i l e d to f i n d m u t a g e n i c i t y f o r l i n o l e i c a c i d hydroperoxide (52). The reason for weak m u t a g e n i c i t y of hydroperoxide i s not c l e a r . I t i s known t h a t f r e e r a d i c a l damage t o n u c l e i c a c i d s c a n be i n d u c e d b y r a d i a t i o n ( 5 3 ) , and DNA r a d i c a l s h a v e b e e n d e t e c t e d a f t e r e x p o s u r e o f DNA t o lipid p e r o x i d a t i o n (54). H o w e v e r , i t may b e e r r o n e o u s t o e x t r a p o l a t e t h e ESR s i g n a l s i n m o d e l s y s t e m s i n t o r e l e v a n c e c o n c e r n i n g i n v i v o DNA d a m a g e w i t h c o n c o m i t a n t m u t a g e n i c i t y .

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch004

2

2

It has been i m p l i e d t h a t secondary p r o d u c t s of lipid a u t o x i d a t i o n are mutagenic. Interest i n t h i s area of research was s t i m u l a t e d when M u k a i a n d G o l d s t e i n ( 5 5 ) , as w e l l as others, reported that malondialdehyde e l i c i t e d a mutagenic response b y t h e Ames test. Since some e v i d e n c e for DNA c r o s s l i n k i n g by malondialdehyde has b e e n shown i n c h e m i c a l models (56), i t m i g h t be presumed t h a t t h i s r e a c t i o n i s the molecular b a s i s of the mutagenicity. However, the importance o f t h e o b s e r v e d r e s p o n s e was q u e s t i o n e d b y M a r n e t t a n d T u t t l e (57), who f o u n d v e r y w e a k m u t a g e n i c i t y w i t h h i g h l y p u r i f i e d malondialdehyde. A c c o r d i n g to them, i m p u r i t i e s from the use of tetraethoxypropane to generate malondialdehyde probably were r e s p o n s i b l e for the greater mutagenicity observed by others. As p o i n t e d o u t i n the t e x t above, l i p i d epoxides are common s e c o n d a r y p r o d u c t s o f a u t o x i d a t i o n . F o r a number o f p o t e n t mutagens, l i k e benzo[or]pyrene, the u l t i m a t e mutagen has been found t o be an e p o x i d e o f t h e p a r e n t compound, w h i c h i n t u r n undergoes n u c l e o p h i l i c s u b s t i t u t i o n by the amino group o f a DNA b a s e p a i r , s u c h a s g u a n i n e ( 5 8 - 6 0 ) . I t has been p o s t u l a t e d b y some w o r k e r s t h a t l i p i d e p o x i d e s a l s o may b e m u t a g e n i c b y a s i m i l a r mechanism. A c o n v i n c i n g m u t a g e n i c r e s p o n s e was n o t o b t a i n e d when e i t h e r c i s - o r t r a n s - 9 , 1 0 - e p o x y o c t a d e c a n o i c acid was i n j e c t e d i n t o m i c e ( 6 1 ) . H o w e v e r , many m u t a g e n i c epoxides have been c h a r a c t e r i z e d as h a v i n g e l e c t r o n - w i t h d r a w i n g s u b s t i t u e n t groups that cause the epoxide t o be more s u s c e p t i b l e to n u c l e o p h i l i c attack (62). F o r t h i s r e a s o n , we t e s t e d b y t h e Ames m e t h o d ( 6 3 ) t h e m u t a g e n i c i t y o f a n u m b e r o f f a t t y epoxides w i t h v i c i n a l f u n c t i o n a l i t y as shown i n F i g u r e 9 . These fatty epoxides were i s o l a t e d from a m i x t u r e o f p r o d u c t s obtained after the free r a d i c a l decomposition of 13-hydroperoxylinoleic a c i d (11). M e t h y l e s t e r s were s y n t h e s i z e d from f a t t y acids with diazomethane. Despite the presence of e l e c t r o n - w i t h d r a w i n g g r o u p s v i c i n a l t o t h e e p o x i d e , m u t a g e n i c i t y was n o t observed even a t t h e 2000 pg l e v e l p e r p l a t e ( 5 1 ) . Apparently, the

132

Control 114

233°

129

130

130

176

by more than LSD.

1

Exceeds ' c o n t r o l

(

by more than LSD

1

144

158

171

235

S-9 Added

experiment.

171

158

164

300°

Exceeds ' c o n t r o l

Each column i n d i c a t e s a separate

164

538°

0

No. S-•9

TA 100

b

38

39

35

55

0

27

26

37

No. S-9

44

41

44

41

46

58

58

94

b

S-9 Added

TA 98

(Methyl E s t e r )

Revertants per p l a t e

f o r Mutagenicity of 13-Hydroperoxylinoleate

50

100

500

1000

pg/plate

Test compound,

Ames Test

Table I

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch004

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch004

GARDNER

Figure

Effects

of Lipid

Hydroperoxides

9. Structures of fatty ester epoxides tested for mutagenicity by the method of Ames et al. (63). (Reproduced with permission from Ref 51.)

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch004

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X E N O B I O T I C S IN F O O D S A N D F E E D S

lack of response stems from t h e 1 , 2 - d i s u b s t i t u t i o n o f t h e e p o x i d e , w h i c h u s u a l l y d i m i n i s h e s t h e response ( 6 2 , 6 4 ) . The s i z e o f t h e h y d r o c a r b o n s i d e c h a i n s a l s o may h a v e a n e f f e c t . Systematic studies of a series of g l y c i d y l ethers indicated t h a t m u t a g e n i c i t y was c o n s i d e r a b l y r e d u c e d when t h e s i d e c h a i n exceeded 4-6 carbons (65). The r o l e o f l i p i d h y d r o p e r o x i d e s i n activating chemical mutagens i s more c o n v i n c i n g . A number o f studies have demonstrated that l i p i d hydroperoxides can i n i t i a t e the free radical oxidation of the carcinogen to the ultimate active form. F o r e x a m p l e , b e n z o [ a ] p y r e n e was o x i d i z e d t o t h e h i g h l y mutagenic 7,8-dihydroxy-9,10-epoxy-7,8,9,10tetrahydrobenzo[a]pyrene i n the presence of 13-hydroperoxylinoleic a c i d and the c a t a l y s t , hematin (66). S i m i l a r l y , F l o y d e t a l . (67) used 1 3 - h y d r o p e r o x y l i n o l e i c acid and h e m a t i n t o a c t i v a t e N-hydroxy-N-acetyl-2-amino-fluorene i n t o the carcinogens, nitrosofluorene and N-acetoxyacetylaminofluorene. T h u s , l i p i d h y d r o p e r o x i d e s may s e r v e a s efficient oxidants of a variety of chemical carcinogens that require o x i d a t i o n to an active form. This area of research appears to be p r o m i s i n g f o r f u t u r e w o r k .

Literature Cited 1. Gardner, H. W. "Autoxidation of Unsaturated Lipids," Chan, H. W.-S., Ed.; Academic:London, in press. 2. Frankel, Ε. N. Prog. Lipid Res. 1980, 19, 1. 3. Porter, Ν. Α.; Lehman, L. S.; Weber, Β. Α.; Smith, K. J . J. Am. Chem. Soc. 1981, 103, 6447. 4. Chan, H. W.-S.; Matthew, J . Α.; Coxon, D. T. J . Chem. Soc. Chem. Commun. 1980, 235. 5. Mihelich, E. D. J . Am. Chem. Soc. 1980, 102, 7141. 6. O'Connor, D. E . ; Mihelich, E. D.; Coleman, M. C. J . Am. Chem. Soc. 1981, 103, 223. 7. Pryor, W. Α.; Stanley, J . P. J . Org. Chem. 1975, 40, 3615. 8. Scheiberle, P.; Grosch, W.; Kexel, H.; Schmidt, H. L. Biochim. Biophys. Acta 1981, 666, 322. 9. Nakano, M.; Takayama, K.; Shimizu, Y . ; Tsuji, Y . ; Inaba, H.; Migita, T. J . Am. Chem. Soc. 1976, 98, 1974. 10. Hamberg, M. Lipids 1975, 10, 87. 11. Gardner, H. W.; Kleiman, R. Biochim. Biophys. Acta 1981, 665, 113. 12. Tappel, A. L. Fed. Proc. Fed. Am. Soc. Exp. Biol. 1973, 32, 1870. 13. Karel, M.; Schaich, K.; Roy, R. B. J . Agric. Food Chem. 1975, 23, 159. 14. Gardner, H. W. J . Agric. Food Chem. 1979, 27, 220. 15. Porkorny, J.; Janicek, G. Nahrung 1975, 19, 911.

4.

GARDNER

16. 17. 18.

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19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43.

Effects of Lipid Hydroperoxides

83

Miquel, J.; Oro, J.; Bensch, K. G . ; Johnson, J . Ε . , Jr., "Free Radicals i n Biology," Pryor, W. Α., E d . ; Academic:New York, 1977, V o l . III, p. 132. Tappel, A. L., "Free Radicals i n Biology," Pryor, W. Α., Ed.; Academic:New York, 1980, V o l . IV, p. 1. Jacks, T. J.; Hensarling, T. P . ; Muller, L. L.; St. Angelo, A. J.; Neucere, N. J. Int. J . Pept. Protein Res. 1982, 20, 149. Funes, J.; Karel, M. Lipids 1981, 16, 347. Leake, L.; Karel, M. J . Food S c i . 1982, 47, 737. Nielsen, H. Lipids 1981, 16, 215. Schaich, K . ; Karel, M. Lipids 1976, 11, 392. Yong, S. H . ; Karel, M. J . Am. Oil. Chem. Soc. 1978, 55, 352. Zirlin, Α.; Karel, M. J. Food S c i . 1969, 34, 160. L i n , J. S.; Olcott, H. S. J . Agric. Food Chem. 1974, 22, 526. Lewis, S. E.; W i l l s , E. D. Biochem. Pharmacol. 1962, 11, 901. Roubal, W. T.; Tappel, A. L . Arch. Biochem. Biophys. 1966, 113, 5. Gardner, H. W.; J u r s i n i c , P. A. Biochim. Biophys. Acta 1981, 665, 100. Finley, J . W.; Wheeler, E. L.; Witt, S. C. J . Agric. Food Chem. 1981, 29, 404. Gardner, H. W.; Kleiman, R.; Weisleder, D . ; Inglett, G. E. L i p i d s , 1977, 12, 655. Gardner, H. W., unpublished data. Yong, S. H . ; Lau, S.; Hsieh, Y.; Karel, Μ., "Autoxidation i n Food and Biological Systems," Simic, M. G . ; Karel Μ., Eds.; Plenum:New York, 1980, p. 237. Sundberg, R. J., "Chemistry of Indoles," Academic Press:New York, 1970, p. 282-315. Saito, I . ; Imuta, M . ; Nakada, Α.; Matsugo, S.; Matsuura, T. Photochem. Photobiol., 1978, 28, 531. Roy, R. B.; Karel, M. J. Food S c i . 1973, 38, 896. Yong, S. H . ; Karel, M. J . Food S c i . 1979, 44, 568. Njaa, L. R . ; Utne, F.; Braekkan, O. R. Nature 1968, 218, 571. Neukom, Η., "Autoxidation i n Food and Biological Systems," Simic, M. G . ; Karel, Μ., Eds.; Plenum:New York, 1980, p. 249. Kikugawa, K . ; Machida, Y.; Kida, M . ; Kurechi, T. Chem. Pharm. Bull., 1981, 29, 3003. Gruger, Ε. Η., Jr.; Tappel, A. L. Lipids 1970, 5, 326. Porter, Ν. Α.; Lehman, L. S.; Khan, J. A. 184th ACS National Meeting, Organic Section 3, Kansas City, September 12-17, 1982. Gardner, H. W.; Eskins, K . ; Grams, G. W.; Inglett, G. E. Lipids 1972, 7, 324. Gardner, H. W., unpublished data.

84

44. 45. 46. 47. 48. 49. 50.

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51. 52. 53. 54. 55. 56. 57. 58. 59.

60. 61. 62. 63. 64. 65. 66. 67.

X E N O B I O T I C S IN F O O D S A N D F E E D S

Kanner, J.; Mendel, H.; Budowski, P. J . Food Sci. 1977, 42, 60. Weber, F . ; Grosch, W. Z. Lebensm. Unters.-Forsch. 1976, 161, 223. Harman, D. Proc. Natl. Acad. Sci. USA 1981, 78, 7124. Pryor, W. A. Ann. N.Y. Acad. Sci. 1982, 393, 1. McBrien, D. C. H.; Slater, T. F . , Eds., "Free Radicals, Lipid Peroxidation and Cancer," Academic:London, 1982. Yamaguchi, T.; Yamashita, Y. Agric. Biol. Chem. 1979, 43, 2225. Yamaguchi, T.; Yamashita, Y. Agric. Biol. Chem. 1980, 44, 1675. Gardner, H. W.; Crawford, C. G.; MacGregor, J . T. Food Chem. Toxicol., in press. Scheutwinkel-Reich, M.; Ingerowski, G.; Stan, H.-J. Lipids 1980, 15, 849. Myers, L. S., J r . , "Free Radicals in Biology," Pryor, W. Α., Ed., Academic:New York, 1980, Vol. IV, p. 94. Fukuzumi, K. Mem. Fac. Eng. Nagoya Univ. 1978, 30, 200. Mukai, F. H.; Goldstein, B. D. Science 1976, 191, 868. Brooks, B. R.; Kalmerth, O. L. Eur. J. Biochem. 1968, 5, 178. Marnett, L. J.; Tuttle, M. A. Cancer Res. 1980, 40, 276. Jeffrey, A. M.; Blobstein, S. H.; Weinstein, I. B.; Beland, F. Α.; Harvey, R. G.; Kasai, H.; Nakanishi, K. Proc. Natl. Acad. Sci. USA 1976, 73, 2311. Jeffrey, A. M.; Jennette, K. W.; Blobstein, S. H.; Weinstein, I. B.; Beland, F. Α.; Harvey, R. G.; Kasai, H.; Miura, I . ; Nakanishi, K. J . Am. Chem. Soc. 1976, 98, 5714. Essigmann, J . M.; Croy, R. G.; Nadzan, A. M.; Busby, W. F . , J r . ; Reinhold, V. N . ; Büchi, G.; Wogan, G. N. Proc. Natl. Acad. Sci. USA 1977, 74, 1870. Swern, D.; Wieder, R.; McDonough, M.; Meranze, D. R.; Shimkin, M. B. Cancer Res. 1970, 30, 1037. Voogd, C. E . ; van der Stel, J . J.; Jacobs, J . J . J . A. A. Mutat. Res. 1981, 89, 269. Ames, Β. N . ; McCann, J.; Yamasaki, E. Mutat. Res. 1975, 31, 347. Wade, D. R.; Airy, S. C.; Sinsheimer, J . E. Mutat. Res. 1978, 58, 217. Thompson, E. D.; Coppinger, W. J.; Piper, C. E . ; McCarroll, N.; Oberly, T. J.; Robinson, D. Mutat. Res. 1981, 90, 213. Dix, Τ. Α.; Marnett, L. J . J . Am. Chem. Soc. 1981, 103, 6744. Floyd, R. Α.; Soong, L. M.; Walker, R. N . ; Stuart, M. Cancer Res. 1976, 36, 2761.

RECEIVED June

28, 1983

5 Some Lipid Oxidation Products as Xenobiotics P. B. ADDIS, A. SAARI CSALLANY, and S. E. KINDOM

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch005

Department of Food Science and Nutrition, University of Minnesota, St. Paul, MN 55108 Lipid oxidation is important in the quality and acceptability of foods and can influence wholesomeness by forming toxins. One such compound, malonaldehyde (MA), has been shown to be toxic, mutagenic, and possibly carcinogenic. Traditionally, MA has been determined by thiobarbituric acid (TBA) test (1-5). However, since TBA reacts with numerous compounds, it should not be used to quantify MA. Recently, a liquid chromatographic method developed in our laboratory to quantify free MA clearly demonstrated extensive overestimation of MA by TBA. Recent studies suggested that atherogenic and perhaps carcinogenic properties previously attributed to cholesterol were the result of contaminating cholesterol oxidation products; their existence in foods is of concern. Malonaldehyde MA, a three carbon dialdehyde, can experience a number of configurâtional m o d i f i c a t i o n s as discussed by Kwon and Watts ( 6 ) . E n o l i z a t i o n of the diketo form may take place. The e n o l i c t a u tomer may f u r t h e r undergo molecular rearrangement into i t s open c i s - , open trans-, or chelated forms. At pH 3 or lower, MA i s chelated and e x i s t s as 3 - h y d r o x y - a c r o l e i n ; above pH 6.5 MA i s completely d i s s o c i a t e d and e x i s t s as an enolate anion. Between pH 3 and 6.5, MA i s an e q u i l i b r i u m mixture of enolate anion and chelated forms. MA ( a l s o malondialdehyde), i s one of the main secondary products of l i p i d o x i d a t i o n . I t forms a pink c o l o r by condensing with 2 moles of TBA ( 7 ) . B i o l o g i c a l S i g n i f i c a n c e of Malonaldehyde The b i o l o g i c a l s i g n i f i c a n c e of MA stems from the f a c t that i t may be formed i n v i v o or i n food products which are then consumed. Kwon and Brown ( 8 ) demonstrated that MA can c r o s s - l i n k

0097-6156/83/0234-0085$06.00/0 © 1983 A m e r i c a n C h e m i c a l S o c i e t y

X E N O B I O T I C S IN

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FOODS AND

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bovine serum albumin to form a s t a b l e complex. Manzel (9) provided evidence that MA can react with ribonuclease r e s u l t i n g i n polymerization and l o s s of enzymatic a c t i v i t y . Brooks and Klamerth (10) reported that g l y o x a l , a s t r u c t u r a l analogue, was t o x i c to human f i b r o b l a s t s i n c e l l c u l t u r e , by i n h i b i t i n g DNA r e p l i c a t i o n . Klamerth and Levinsky (11) examined r a t s which were fed MA and observed damaged l i v e r DNA and l o s s of template activity. Studies on l i p i d o x i d a t i o n i n v i v o have been c l o s e l y l i n k e d with aging and MA appears to have a r o l e . Chio and Tappel (12) reported that MA takes part i n formation of l i p o f u s c i n "age" pigment production. B i r d and Draper (13) r e c e n t l y studied b i o l o g i c a l e f f e c t s of MA on growth, morphology and macromolecular biosynthesis i n a neonatal r a t s k i n f i b r o b l a s t c e l l c u l t u r e . Acetaldehyde was used as a reference compound. C e l l s exposed to 10~3 M MA f o r 120 hours e x h i b i t e d a l t e r e d morphology, cytoplasmic v a c u o l i z a t i o n , k a r y o r r h e x i s , micro- and m u l t i - n u c l e a t i o n , and a marked reduction i n m i t o t i c index, and DNA-, RNA-, and p r o t e i n - s y n t h e s i z i n g capa­ city. At 10"^ M, MA caused m i t o t i c a b e r r a t i o n s , nuclear morpho­ l o g i c a l i r r e g u l a r i t i e s , a reduced m i t o t i c index and i n h i b i t i o n of RNA and DNA s y n t h e s i s . At 10" and 10"* M, MA induced only f o r ­ mation of small and i r r e g u l a r n u c l e i . MA was approximately 10 times more t o x i c to rat s k i n f i b r o b l a s t s than acetaldehyde. Neither MA or acetaldehyde exerted any n o t i c e a b l e e f f e c t s on c e l l u l a r metabolism, i n d i c a t i n g that s k i n f i b r o b l a s t s are e i t h e r able to c a t a b o l i z e the two aldehydes e f f i c i e n t l y at these con­ c e n t r a t i o n s or are capable of r e p a i r i n g any damage induced at the molecular l e v e l . Subsequently, B i r d et a l . (14) continued t h e i r t o x i c o l o g i c a l study of MA and acetaldehyde using the f i b r o b l a s t system and noted dose-dependent production of micronuclei f o r concentrations of MA between 10"^ and 10"" ^ M. Twelve hours of treatment with MA r e s u l t e d i n chromosomal a b e r r a t i o n s . MA was again about ten times as potent as acetaldehyde with respect to micronuclei formation. MA probably exerts i t s chromosomedamaging e f f e c t s by c r o s s - l i n k i n g strands of DNA, thus producing i n a c t i v a t i n g a l t e r a t i o n s i n DNA s t r u c t u r e which i n h i b i t DNA r e p l i c a t i o n (unless eliminated by r e p a i r ) . Shamberger et a l . (15) observed that MA was carcinogenic to mouse s k i n . Mukai and G o l d s t e i n (16) reported that MA was mutagenic i n h i s t i d i n e - r e q u i r i n g s t r a i n s of Salmonella t y p h i ­ murium. Yau (17) found that MA was h i g h l y mutagenic and cyto­ t o x i c i n mammalian c e l l s and therefore may be a potent carcinogen i n humans. Exposure to as l i t t l e as 20 μΜ MA was c y t o t o x i c and increased mutation frequency among s u r v i v o r s of murine L5178Y lymphoma c e l l c u l t u r e s as w e l l . From the foregoing s t u d i e s , i t i s p o s s i b l e to conclude that MA i s h i g h l y mutagenic, carcinogenic and c y t o t o x i c . However, Marnett and T u t t l e (18) suggested that perhaps the causative agent f o r the mutagenic and carcinogenic p r o p e r t i e s of MA i s an 5

6

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5.

ADDIS E T A L .

Lipid

Oxidation

Products

as

Xenobiotics

87

intermediate formed i n production of MA from 1,1,3,3-tetraethoxypropane (TEP), an intermediate which does not a r i s e during l i p i d p e r o x i d a t i o n . Marnett and T u t t l e (18) noted that 3-ethoxyacrol e i n , an incomplete h y d r o l y s i s product of TEP, was 20 times more mutagenic than MA. When producing MA by h y d r o l y s i s of 1,1,3,3tetramethoxypropane (TMP), both intermediates formed, 3,3-dimethoxy-propanaldehyde and 3-methoxyacrolein, were more mutagenic than MA and they appeared to be more mutagenic than 3-ethoxyacrol e i n as w e l l . Since these intermediates are much stronger mutagens than MA, they could be r e s p o n s i b l e f o r a p o r t i o n of the mutagenicity and c a r c i n o g e n i c i t y a t t r i b u t e d to MA by Shamberger (15) and by Mukai and G o l d s t e i n (16). However, t h i s question i s not yet completely r e s o l v e d . Malonaldehyde Measurements i n Food Products The f i r s t extensive i n v e s t i g a t i o n i n t o the use of TEP and TMP as MA standards f o r the TBA method was conducted by Gutteridge (19). His f i n d i n g s were as f o l l o w s : (1) TEP was hydrolyzed completely i n 90 minutes at 56°C. (2) TMP r e q u i r e d four hours f o r complete h y d r o l y s i s . By that time a 60 ninole TMP s o l u t i o n had l o s t 50% of i t s TBA r e a c t i v i t y . (3) T h i n - l a y e r chromatography showed at l e a s t nine compounds can be recovered from TEP and TMP, a l l of which react with TBA to give a red pigment with maximum absorbance at 532 nanometers. Gutteridge (19) concluded from t h i n - l a y e r chromatography data that bands i n e x t r a c t s from a u t o x i d i z e d polyunsaturated f a t t y acids which react with TBA are not merely polymers of MA; they are "probably l a r g e r molecular weight precursors of MA that are broken down to MA when heated with the TBA reagent." He f u r t h e r suggested that the best method f o r preparing a standard curve from TEP would be to heat TBA d i r e c t l y with TEP without any p r e h y d r o l y s i s , so that p o l y m e r i z a t i o n of MA and l o s s of TBA r e a c t i v i t y could be minimized. As e a r l y as 1951 (20), and numerous times since then, TBA has been used to measure MA l e v e l s i n foods. However, TBA should be used to measure extent of l i p i d o x i d a t i o n i n general, not to q u a n t i f y MA s p e c i f i c a l l y . At the present time there are numerous substances known to i n t e r f e r e with the TBA r e a c t i o n . Dugan (21) showed that sucrose and some compounds i n wood smoke react with TBA to produce a red c o l o r . A yellow pigment found by Yu and Sinnhuber (22) was able to s i g n i f i c a n t l y a f f e c t the TBA method; however, i t could be separated by chromatography. Saslaw et a l . (23) reported that other u n i d e n t i f i e d carbonyl compounds w i l l react with TBA. A d d i t i o n a l l y i t was p o s s i b l e (24) by i r r a d i a t i o n to produce TBA r e a c t i v e substances, none of which were MA. Kwon and O l c o t t (25) concluded that the TBA method was a q u a n t i t a t i v e measure of l i p i d o x i d a t i o n , but only during i n i t i a l stages of o x i d a t i o n since h i g h l y polymerized MA ( u n r e a c t i v e ) forms i n advanced stages of o x i d a t i o n . Schneir et a l . (26) i n d i c a t e d that N-acetyl-neuraminic a c i d has a high absorbance at 549 nm i n the TBA t e s t . Marcuse and Johansson (27) i d e n t i f i e d

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch005

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X E N O B I O T I C S IN

FOODS A N D

FEEDS

o t h e r a l d e h y d e s w h i c h p r o d u c e d a p o s i t i v e TBA r e a c t i o n , i n c l u d i n g 2 , 4 - a l k a d i e n a l s and 2 - a l k e n a l s . Baumgartner et a l . (28) noticed t h a t a c e t a l d e h y d e i n t h e p r e s e n c e o f s u c r o s e a n d a c i d was c a p a b l e of r e a c t i n g w i t h TBA. P r y o r e t a l . ( 2 9 ) d e t e r m i n e d t h a t MA may be g e n e r a t e d f r o m i t s p r o s t a g l a n d i n - l i k e e n d o p e r o x i d e precursors by t h e c o n d i t i o n s ( a c i d and h e a t ) o f t h e TBA p r o c e d u r e . Ohkawa e t a l . (30) n o t e d t h a t g l u c o s e , s u c r o s e and N - a c e t y l - n e u r a m i n i c a c i d c o u l d i n t e r f e r e w i t h MA d e t e r m i n a t i o n b y T B A . I n recent s t u d i e s ( u n p u b l i s h e d ) i n our l a b o r a t o r y , copper was f o u n d to d i m i n i s h c o l o r d e v e l o p m e n t i n the TBA t e s t . The f o r e g o i n g s t u d i e s l e d W i t t e e t a l . (5) and l a t e r R e t h w i l l et a l . (31) t o m o d i f y t h e TBA p r o c e d u r e of T a r l a d g i s e t a l . (4) i n o r d e r t o e l i m i n a t e h e a t i n g and d i s t i l l a t i o n . I n most c a s e s T B A - i n t e r f e r e n c e s r e s u l t i n g from o t h e r compounds have l e a d to elevated l e v e l s q u o t e d f o r MA. T h e n e e d f o r a d i r e c t m e t h o d o f MA determination is obvious. R e c e n t l y , C s a l l a n y and c o w o r k e r s (32) developed a h i g h performance l i q u i d c h r o m a t o g r a p h i c (HPLC) m e t h o d f o r q u a n t i f i c a t i o n o f f r e e MA i n t i s s u e s o r m e a t . HPLC s e p a r a t i o n w a s p e r f o r m e d w i t h a T S K G 1 0 0 0 PW c o l u m n u s i n g a m o b i l e p h a s e o f 0 . 1 M N a 3 P 0 4 , pH 8 . 0 b u f f e r a t a f l o w r a t e o f 0.6 ml per minute. T h e e l u a n t was m o n i t o r e d a t 267 nm. Free MA i n a t i s s u e s a m p l e c a n b e s e p a r a t e d a n d q u a n t i f i e d i n a p p r o x i m a t e l y 50 m i n u t e s a t l e v e l s as l o w as one n a n o g r a m p e r i n j e c tion. T a b l e I o u t l i n e s some r e s u l t s t h a t w e r e o b t a i n e d o n MA c o n t e n t of food. TABLE

I.

C o m p a r i s o n of MA

Sample B e e f (N =

9)

SD Pork

(N

=

9)

SD Pg/g w e t

MA c o n t e n t

of

c o n t e n t (Pg/g) HPLC TBA 0.14 0.44

meat

by HPLC a n d TBA

TBA/HPLC r a t i o 3.1

0.085

0.19

2.2

0.11

0.39

3.5

0.06

0.12

2.0

tissue

C o m p a r i s o n s w e r e made w i t h T B A . A s c a n be s e e n , T B A g r e a t l y o v e r e s t i m a t e s t h e t r u e a m o u n t o f f r e e MA w h i c h i s p r e s e n t , l e a d i n g the a u t h o r s (32) t o c o n c l u d e t h a t i t i s not an a c c e p t a b l e m e a n s o f d e t e r m i n i n g MA l e v e l s i n f o o d p r o d u c t s o r t i s s u e s . A more a c c u r a t e d e s c r i p t i o n o f w h a t t h e t e s t m e a s u r e s w o u l d be "TBA-reactive substances." I n s p i t e of t h i s f a c t , numerous a u t h o r s h a v e r e p o r t e d l e v e l s o f MA u s i n g t h e T B A p r o c e d u r e i n the l i t e r a t u r e ( c f . 33,34). The v a r i a b i l i t y of t h e TBA p r o c e d u r e i s a l s o much g r e a t e r t h a n t h a t f o r t h e HPLC d e t e r m i n a t i o n o f MA ( T a b l e I).

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch005

5.

ADDIS E T A L .

Lipid

Oxidation

Products

as

Xenobiotics

89

C h o l e s t e r o l O x i d a t i o n Products As i s the case f o r MA, there i s l i t t l e agreement concerning e x i s t e n c e and s i g n i f i c a n c e of c h o l e s t e r o l o x i d a t i o n products i n foods. The r o l e of c h o l e s t e r o l c o n t a i n i n g foods i n the American d i e t has been the subject of many i n v e s t i g a t i o n s i n recent years, l a r g e l y due to hypothesized l i n k s between c h o l e s t e r o l and coro­ nary heart disease (CHD) i n humans. I t i s not the purpose of t h i s paper to review a l l of the pros and cons concerning l i p i d involvement i n CHD. However, a few of the h i g h l i g h t s as they p e r t a i n to c h o l e s t e r o l are worth reviewing. The o r i g i n a l studies l i n k i n g c h o l e s t e r o l to CHD were performed on v e g e t a r i a n animals which were fed extremely high d i e t a r y l e v e l s of c h o l e s t e r o l . These types of studies were c r i t i c i z e d f o r t h e i r choice of animal and l e v e l s of c h o l e s t e r o l f e d . From another standpoint, T a y l o r and coworkers (35) have s e r i o u s l y questioned the v a l i d i t y of many e a r l y experiments based on the s u p p o s i t i o n that the c h o l e s t e r o l used was most l i k e l y contaminated with o x i d a t i o n products of c h o l e s t e r o l and that these o x i d a t i o n products, not c h o l e s t e r o l , were the cause of a t h e r o s c l e r o s i s . Therefore, c h o l e s t e r o l may be much s a f e r than believed by o r g a n i z a t i o n s such as the American Heart A s s o c i a t i o n which recommends reducing the amount of cho­ l e s t e r o l i n our d i e t s . On the other hand, r e p o r t s i n d i c a t i n g that c h o l e s t e r o l o x i d a t i o n products are present i n c e r t a i n types of foods r a i s e s questions about t h e i r s a f e t y . I n 1963, c h o l e s t e r o l hydroperoxides were reported ( 3 6 ) i n egg-containing foods i r r a d i a t e d by s u n l i g h t . Chicoye et a l . (37) observed the f o l l o w i n g 5 p h o t o x i d a t i o n d e r i v a t i v e s of c h o l e s t e r o l i n s p r a y - d r i e d y o l k exposed to e i t h e r 40-watt f l u o r e s c e n t lamp (approx. 280 hours) or summer s u n l i g h t (5 hours): 33~hydroxycholest-5-en-7-one (7-keto); c h o l e s t - 5 - e n e - 3 3 , 7 a - d i o l ( 7 a - d i o l ) ; cholest-5-ene-33,73-diol ( 7 3 - d i o l ) ; 5,63-epoxy-5a-cholestan-33-ol (3-epoxide) and 5a-cholestane-33,5a , 6 3 - t r i o l ( t r i o l ) . Subse­ quently, T s a i et a l . (38) developed methodology to demonstrate the presence of 5,6a-epoxy-5a-choiestan-33-ol (α-epoxide) i n d r i e d egg products which were spiked with the epoxides. Refined e d i b l e beef tallow used as a deep-fat f r y i n g medium by f a s t - f o o d r e s t a u r a n t s can be subjected to c o n d i t i o n s which are s u i t a b l e f o r o x i d a t i o n of c h o l e s t e r o l . Ryan et a l . (39) i d e n t i f i e d s e v e r a l c h o l e s t e r o l o x i d a t i o n products i n t a l l o w heated at 180°C i n t e r m i t t e n t l y (8 hours/day) during storage times of 75 and 150 hours. The extent to which these oxides are absorbed by foods during f r y i n g i s unknown. The i n h i b i t i o n of c h o l e s t e r o l o x i d a t i o n and u l t i m a t e improvement of food safety may now be an i n t e r e s t i n g area of study. Atherogenic!ty O x i d a t i o n products derived from c h o l e s t e r o l appear to have the g r e a t e s t p o t e n t i a l f o r h e a l t h impairment of a l l c l a s s e s of compounds i s o l a t e d from r a n c i d foods up to the present time. Potent a n g i o t o x i c e f f e c t s have been noted (40-44) f o r s e v e r a l of these compounds leading researchers to hypothesize a l i k e l y r o l e

90

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Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch005

f o r them i n CHD. The e a r l y work of S e i f t e r and B a e d e r (43) provides clues which suggest that the atherogenic effects a t t r i b u t e d t o c h o l e s t e r o l may h a v e b e e n d u e t o i t s c o n t a m i n a t i n g oxidation products. The s t u d y i n v o l v e d i n d u c i n g h y p e r c h o l e s t e r ­ o l e m i a by d i e t a r y and endogenous ( h o r m o n a l ) mechanisms. Hormonal treatment l e d to severe h y p e r c h o l e s t e r o l e m i a and y e t l e s s atherog e n i c i t y compared t o t h e m i l d e r h y p e r c h o l e s t e r o l e m i a p r o d u c e d by diet. I n r e t r o s p e c t , i t seems l o g i c a l t h a t d i e t a r y i n d u c t i o n of h y p e r c h o l e s t e r o l e m i a i n c l u d e d t h e i n t r o d u c t i o n of cholesterol oxidation products into experimental animals. I n c o n t r a s t , one w o u l d e x p e c t e n d o g e n o u s h y p e r c h o l e s t e r o l e m i a t o be l i m i t e d t o n a t i v e c h o l e s t e r o l , f r e e of o x i d a t i o n p r o d u c t s . I n 1 9 7 1 , i t was s u g g e s t e d (44) t h a t t h e l e v e l of α - e p o x i d e i n h u m a n s e r u m may b e r e l a t e d t o t h e s e v e r i t y o f atherosclerosis. T h i s h y p o t h e s i s was b a s e d o n t h e m e a s u r e m e n t o f v e r y h i g h c o n c e n ­ t r a t i o n s ( 2 5 0 - 3 , 2 5 0 yg/100 m l serum) o f α-epoxide i n Type I I h y p e r c h o l e s t e r o l e m i a p a t i e n t s , whereas c o n t r o l s c o n t a i n e d l e s s t h a n 5 yg/100 m l serum. Imai et a l . (40) demonstrated angiotoxic e f f e c t s from contaminants of USP-grade c h o l e s t e r o l . By t h e u s e of m e t h a n o l i c e x t r a c t i o n , U S P - g r a d e c h o l e s t e r o l was p u r i f i e d a n d the o x i d a t i o n products concentrated. Both newly purchased and 5 year o l d c h o l e s t e r o l were e x t r a c t e d . P u r i f i e d c h o l e s t e r o l was o b t a i n e d by t h e d i b r o m i n a t i o n p r o c e d u r e a c c o r d i n g to F i e s e r ( 4 5 ) . R a b b i t s were a d m i n i s t e r e d c o n c e n t r a t e of o x i d a t i o n p r o d u c t s o r p u r i f i e d c h o l e s t e r o l by g a v a g e , s a c r i f i c e d , and t h e i r a o r t a s examined m i c r o s c o p i c a l l y f o r a n g i o t o x i c e f f e c t s . The c o n c e n ­ t r a t e , c o n t a i n i n g p r o d u c t s of spontaneous o x i d a t i o n of c h o l e s ­ t e r o l , i n c r e a s e d t h e f r e q u e n c y of dead a o r t i c smooth muscle c e l l s a n d i n d u c e d f o c a l i n t i m a i edema i n r a b b i t s 24 h o u r s after gavage. B o t h new a n d o l d c h o l e s t e r o l e x t r a c t s w e r e f o u n d t o b e angiotoxic. O l d c h o l e s t e r o l , new c h o l e s t e r o l a n d c o n t r o l s ( g e l a t i n ) showed t h e f o l l o w i n g f r e q u e n c y of a g g r e g a t e d e b r i s and d e g e n e r a t e d c e l l s p e r 100 n u c l e a t e d c e l l s : 0 . 7 , 0 . 2 , 0.03 and 7 . 6 , 4 . 4 a n d 0 . 6 1 . P u r i f i e d c h o l e s t e r o l a p p e a r e d t o h a v e no s u c h effect. I n a longer term study, c h o l e s t e r o l o x i d a t i o n product " c o n c e n t r a t e " was a d m i n i s t e r e d 1 g/kg body w e i g h t p e r 7 week period. I n t i m a i d i f f u s e f i b r o u s l e s i o n s w i t h o u t foam c e l l s o r h y p e r c h o l e s t e r o l e m i a were i n d u c e d . P u r i f i e d c h o l e s t e r o l at the same d o s e h a d n o e f f e c t . I n a s e q u e l , Peng e t a l . (41) p u r i f i e d known o x i d a t i o n p r o d u c t s of c h o l e s t e r o l and s t u d i e d t h e i r t o x i c i t y i n c u l t u r e d r a b b i t a o r t i c smooth muscle c e l l s . Using thin layer chromato­ g r a p h y f o r s e p a r a t i o n f o l l o w e d b y UV d e t e c t i o n i n 5 0 % a q u e o u s s u l f u r i c a c i d spray, the o x i d a t i o n products separated into 6 f r a c t i o n s a c c o r d i n g to t h e i r m o b i l i t i e s and were used i n the c e l l culture. P o t e n t c y t o x i c e f f e c t s were n o t e d f o r a number of t h e identified cholesterol oxidation products. In contrast, purified c h o l e s t e r o l a t t h e same c o n c e n t r a t i o n p r o d u c e d n o t o x i c effects. The r e s u l t s d e m o n s t r a t e d t h a t 2 5 - h y d r o x y c h o l e s t e r o l and t r i o l w e r e t h e most t o x i c a g e n t s . Peng e t a l . (42) confirmed and

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch005

5.

ADDIS E T

AL.

Lipid

Oxidation

Products

as

Xenobiotics

91

extended t h e i r e a r l i e r work on a n g i o t o x i c i t y of c h o l e s t e r o l o x i d a t i o n products. Commercially obtained c h o l e s t e r o l o x i d a t i o n products were introduced i n t o r a b b i t a o r t i c smooth muscle c e l l c u l t u r e s . Degree of c y t o x i c i t y was estimated as a percentage of dying and dead c e l l s i n c u l t u r e s w i t h i n 24 hours of a p p l i c a t i o n . The r e s u l t s i n d i c a t e d that 25-hydroxycholesterol and t r i o l were the most t o x i c compounds t e s t e d . A d d i t i o n a l studies i n d i c a t e d that when these o x i d a t i o n d e r i v a t i v e s were added to c u l t u r e d c e l l s they s i g n i f i c a n t l y depressed the a c t i v i t y of 3-hydroxy-3m e t h y l g l u t a r y l coenzyme A reductase, a regulatory enzyme of c h o l e s t e r o l b i o s y n t h e s i s . The degree of i n h i b i t i o n by 25-hyd r o x y c h o l e s t e r o l was remarkable: 3 yg/inl i n c u l t u r e r e s u l t e d i n an 83% i n h i b i t i o n . P u r i f i e d c h o l e s t e r o l showed no c y t o t o x i c e f f e c t s and minimal i n h i b i t i o n of c h o l e s t e r o l b i o s y n t h e s i s . Most r e c e n t l y , Peng et a l . (46) has demonstrated that i n monkeys, 24 hours a f t e r i n g e s t i o n of ^C-25-hydroxycholesterol, 55.1%, 34.7% and 10.2% of the r a d i o a c t i v i t y was located i n low density l i p o p r o t e i n s (LDL), very low density l i p o p r o t e i n s (VLDL) and high d e n s i t y l i p o p r o t e i n s (HDL), r e s p e c t i v e l y . This i s i n contrast to l ^ C - c h o l e s t e r o l fed c o n t r o l s a f t e r the same time period which revealed the f o l l o w i n g d i s t r i b u t i o n of l a b e l : LDL (47.6%), VLDL (3.1%) and HDL (49.3%). Since the ultimate metabolic f a t e of VLDL i s conversion to LDL which i s then transported to p e r i p h e r a l t i s s u e s , i t was speculated that p r o p o r t i o n a l l y more 25-hydroxyc h o l e s t e r o l would be incorporated i n t o vascular t i s s u e where i t could i n h i b i t c h o l e s t e r o l b i o s y n t h e s i s , cause membrane dysfunct i o n and induce a r t e r i a l i n j u r y . Smith (47) and Simic and K a r e l (48) have contributed u s e f u l monographs on the subjects of l i p i d ( i n c l u d i n g c h o l e s t e r o l ) o x i d a t i o n i n food and b i o l o g i c a l systems. C a r c i n o g e n i c i t y of C h o l e s t e r o l Oxides: H i s t o r i c a l Background S p e c u l a t i o n i n t o the carcinogenic p o t e n t i a l of c h o l e s t e r o l and i t s d e r i v a t i v e s began once t h e i r s t r u c t u r a l r e l a t i o n s h i p to p o l y c y c l i c hydrocarbons was r e a l i z e d . In 1933, Roffo (49) suggested that photo-induced o x i d a t i o n products of c h o l e s t e r o l might be responsible f o r UV-induced carcinogenesis of the s k i n . About t h i s same time, i t was recognized (50) that a r t i f a c t s ( c h o l e s t e r o l o x i d a t i o n products) may a r i s e during the bromination-debromination r e a c t i o n (51) used i n p u r i f i c a t i o n of cholest e r o l . A l s o , when Haslewood (52) showed t r i o l to be present i n o x - l i v e r residue, i t was not p o s s i b l e to decide whether these compounds are n a t u r a l c o n s t i t u e n t s or a u t o x i d a t i o n products formed during i s o l a t i o n (53). B i s c h o f f and Rupp (54) found a 32% l o c a l incidence of cancer i n ovariectomized mice administered a crude progesterone preparation ( c o n t a i n i n g 20 mg/mouse of u n i d e n t i f i e d c h o l e s t e r o l o x i d a t i o n products) subcutaneously i n sesame o i l verses 0% f o r pure progesterone. F i e s e r (55) postul a t e d that a carcinogenic conversion product of c h o l e s t e r o l was the offender. The foregoing f i n d i n g s coupled by the ease with which c h o l e s t e r o l i s o x i d i z e d i n aqueous c o l l o i d a l s o l u t i o n s by i

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch005

92

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a i r (56,57), a c o n d i t i o n that conceivably could be d u p l i c a t e d i n v i v o , prompted a f u r t h e r i n v e s t i g a t i o n (58). Problem of O i l y V e h i c l e P u r i f i e d c h o l e s t e r o l , a l l known i n i t i a l and subsequent o x i d a t i o n products of c h o l e s t e r o l and some r e l a t e d compounds were tested: cholest-5-en-3-one; 7 a - d i o l ; 7 3 - d i o l ; 7-keto; cholesta-3,5-dien-7-one; α-epoxide; t r i o l ; choiest-4-en-3-one; 63-hydroperoxychoiest-4-en-3-one; 63-hydroxycholest-4-en-3-one; cholest-4-ene-3,6-dione; choiest-5-ene-33,43-diol; 5a-cholest-6ene-33,5a-diol; 53-cholestan-33-ol; cholesta-1,4-dien-3-one; 5a-cholest-7-en-33-ol; choiesta-7,9-dien-33-ol acetate; 7,8,9,11d i e p o x y - 2 2 - i s o a l l o s p i r o s t a n - 3 3 - o l acetate; c h o l e s t e r y l acetoacet a t e ; and c h o l e s t e r y l i s o h e p t y l a t e . Many were carcinogenic when administered subcutaneously i n t o Marsh-Buffalo mice with sesame o i l as the v e h i c l e ; α-epoxide, 63-hydroperoxycholest-4-en-3-one and choiest-4-en-3,6-dione were most potent (34-66% i n c i d e n c e of fibrosarcoma verses 1.4% f o r v e h i c l e c o n t r o l ) . A l l share the property of oxygen linkage at carbon 6. 63-hydroxychoiest4-en-3-one was m i l d l y (15%) c a r c i n o g e n i c . Sesame o i l was described (58) a cocarcinogen because negative r e s u l t s were obtained i f c e r t a i n of these carcinogens were given as aqueous colloids. Some s t e r o i d s that are normal body c o n s t i t u e n t s were c a r c i n o g e n i c i f i n j e c t e d i n sesame o i l and heated or oxidized o i l had p o s i t i v e tumorigenic r e s u l t s when administered alone. Hieger (59,60), on the other hand, c o n c l u s i v e l y stated a carcinogenic e f f e c t f o r c h o l e s t e r o l subcutaneously i n j e c t e d i n an o i l y v e h i c l e . B i s c h o f f and Bryson (61) were able to show that the c r i t i c a l d i f f e r e n c e between these r e s u l t s was the p h y s i c a l s t a t e of c h o l e s t e r o l at the i n j e c t i o n s i t e . H i e g e r s method had produced c r y s t a l s at the dose s i t e and was an example of smoothsurface c a r c i n o g e n e s i s . To prevent t h i s , c h o l e s t e r o l concentra­ t i o n i n an o i l must be below 4%. C h o l e s t e r o l o x i d a t i o n product e v a l u a t i o n has continued. However, a myriad of problems have plagued these i n v e s t i g a t i o n s i n c l u d i n g i n s u f f i c i e n t s u r v i v a l time among experimental animals, spontaneous occurrence of fibrosarcoma and membrane encapsulation (oleoma) of i n j e c t e d m a t e r i a l , probably due to p h y s i c a l ( s u r f a c e or texture) e f f e c t s (62-65). Frequently studies were complicated by the use of an o i l y v e h i c l e , as discussed p r e v i o u s l y f o r sesame oil. I t was suggested that o i l s not be used as c a r r i e r s due to t h e i r v a r i a b i l i t y , c a p a c i t y to react m e t a b o l i c a l l y with s t e r o i d s , a b i l i t y to promote c r y s t a l l i z a t i o n of c h o l e s t e r o l d e r i v a t i v e s , p o t e n t i a l f o r c o n t a i n i n g other carcinogen contaminants and a b i l i t y to induce t i s s u e inflammation (66). The only product of c h o l e s t e r o l o x i d a t i o n known to be c a r ­ cinogenic i n the absence of o i l i s α-epoxide, which was shown to be c a r c i n o g e n i c i n r a t s and mice (62,63). U l t r a v i o l e t - I n d u c e d Skin Carcinogenesis Formation of polar c h o l e s t e r o l d e r i v a t i v e s was demonstrated (67,68) i n human and h a i r l e s s mouse s k i n specimens i r r a d i a t e d i n 1

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u l t r a v i o l e t (UV) l i g h t . I t was c l e a r l y e s t a b l i s h e d that α-epoxide o r i g i n a t e d from n a t u r a l l y o c c u r r i n g s t e r o l s (68). S k i n of h a i r l e s s mice subjected to chronic low l e v e l s of UV d i s p l a y e d increased epoxide at 4 weeks and maximum l e v e l s at 10 weeks (69,70). Subsequently, i t was shown α-epoxide hydrase increased s t a r t i n g at 8 and peaking at 15 weeks and that i t s e l e v a t i o n c o i n c i d e d with a d e c l i n e i n epoxide but also a rapid increase i n tumor incidence (70,71). D i e t a r y intake of ascorbate, butylated hydroxytoluene (BHT), dl-α-tocopherol and reduced g l u t a t h i o n e decreased l e v e l s of α-epoxide by 50% and suppressed tumor formation induced by UV l i g h t (72,73). Although the c i t e d evidence argues f o r α-epoxide as a car­ cinogen, no data e x i s t s demonstrating i t as a t o p i c a l carcinogen (74). Colon Carcinogenesis The colon i s the most common s i t e f o r development of cancer i n the U.S. population. A s p e c i f i c carcinogen has not been i d e n t i f i e d , but a potent mutagen has r e c e n t l y been i s o l a t e d from feces of h i g h - r i s k p a t i e n t s (75). E p i d e m i o l o g i c a l studies suggest that d i e t s high i n animal f a t and p r o t e i n , e s p e c i a l l y beef, and low i n n a t u r a l carbohydrate, i n c l u d i n g f i b e r , may be i n v o l v e d i n the e t i o l o g y (76,77) and i t has been reported that f e c a l mutagenic a c t i v i t y i s higher i n subjects consuming such d i e t s (78). The fact that ascorbate or tocopherol supplemen­ t a t i o n reduced f e c a l mutagenic a c t i v i t y suggests need f o r studies on the p o s s i b l e r o l e of l i p i d o x i d a t i o n products (79). A p o s s i b l e r o l e i n colon cancer f o r c h o l e s t e r o l and i t s o x i ­ d a t i o n products i s suggested by s e v e r a l studies (76,80-83). Hill and coworkers (81,84) observed that a high f a t d i e t reduced i n t e s t i n a l redox p o t e n t i a l and increased l e v e l s of f e c a l cho­ l e s t e r o l and b i l e a c i d metabolites. P a t i e n t s with adenomatous polyps and u l c e r a t i v e c o l i t i s , both at increased r i s k from colon cancer (86,87), or with a c t i v e bowel cancer, excreted greater l e v e l s of c h o l e s t e r o l , coprostanol and t r i o l than c o n t r o l s (80,85). Reddy and coworkers (88-90) evaluated c a r c i n o g e n i c or tumorpromoting a c t i v i t y of c h o l e s t e r o l , α-epoxide, t r i o l and primary and secondary b i l e a c i d s , with and without i n d u c t i o n by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Tumor development r e q u i r e d MNNG, and b i l e a c i d s acted as promoters. Cholesterol and i t s metabolites were not found to have promoting a c t i v i t y . Other Cancers C h o l e s t e r o l , a- and 3-epoxides were present i n 45% of breast f l u i d specimens examined with α-isomer predominating. Epoxide l e v e l s tended to be higher i n f l u i d s with high c h o l e s t e r o l l e v e l s and i n those taken from o l d e r women prompting researchers to speculate that benign breast disease and cancer could r e s u l t (91). The n o n l a c t a t i n g breast concentrates i t s s e c r e t i o n s and thereby exposes the mammary e p i t h e l i u m to α-epoxide. Schaffner e t a l . (92) noted high l e v e l s of c h o l e s t e r o l

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epoxides i n p r o s t a t i c s e c r e t i o n s and postulated a r o l e f o r them i n p r o s t a t i c cancer. Recently, Smith et a l . (93) demonstrated that air-aged com­ m e r c i a l samples of USP- o r reagent-grade c h o l e s t e r o l contain com­ pounds which are mutagenic toward three s t r a i n s of Salmonella typhimurum. Future Research Needs. I t i s obvious that studies on t o x i ­ c i t y and c a r c i n o g e n i c i t y of l i p i d o x i d a t i o n products, as they occur i n food, are i n t h e i r infancy. I t i s s t i l l not e s t a b l i s h e d that any of these compounds c o n s t i t u t e a threat to p u b l i c h e a l t h . I t i s c e r t a i n that, a t the l e v e l s normally encountered i n foods, acute e f f e c t s on humans would not be seen. The chronic area remains an enigma. I t i s known that l e v e l s of MA i n foods have been overstated by the TBA t e s t . Extensive studies are urgently needed to quantify f r e e MA d i r e c t l y by HPLC (32). Further i n v e s t i g a t i o n s are also necessary to determine more f u l l y the t o x i c i t y of MA. Even l e s s w e l l understood than MA i s the p o s s i b l e occurrence of c h o l e s t e r o l o x i d a t i o n products i n foods and t h e i r health s i g n i f i c a n c e . I t seems c l e a r that some of the products of cho­ l e s t e r o l a u t o x i d a t i o n are atherogenic. Much more research w i l l be required to e s t a b l i s h or refute t h e i r proposed carcinogenic p r o p e r t i e s . Methods f o r the determination of c h o l e s t e r o l oxida­ t i o n products i n foods and studies to e s t a b l i s h l e v e l s of occur­ rence, i f any, are most urgently needed. Further studies on a n t i o x i d a n t s and procedures f o r the i n h i b i t i o n of o x i d a t i o n are a l s o needed. I t i s d i f f i c u l t to overstate the p o t e n t i a l impor­ tance to the animal products industry of studies on q u a n t i f i ­ c a t i o n of c h o l e s t e r o l oxides i n food products. Concern about t h i s area of research i s becoming widespread (47,48). Acknowledgments S c i e n t i f i c J o u r n a l S e r i e s Paper No. 13,492, Minnesota A g r i c u l t u r a l Experiment S t a t i o n .

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Taylor, C. B.; Peng, S-K.; Werthessen, N. T.; Tham, P.; Lee, Κ. T. Am. J . Clin. Nutr. 1979, 32, 40-57. Acker, L.; Greve, H. Fette Seif. Anstrich. 1963, 1009. Chicoye, E.; Powrie, W. D.; Fennema, O. J. Food Sci. 1968, 33, 581-7. Tsai, L. S.; Ijichi, K.; Hudson, C. Α.; Meehan, J . J . Lipids 1980, 15, 124-8. Ryan, T. C.; Gary, J . I.; Morton, I. D. J . Sci. Food Agric. 1981, 32, 305-8. Imai, H.; Werthessen, N. T.; Taylor, C. B.; Lee, Κ. T. Arch. Path. Lab. Med. 1976, 100, 565-72. Peng, S. K.; Taylor, C. B.; Tham, P.; Werthessen, N. T.; Mikkelson, B. Arch. Path. Lab. Med. 1978, 102, 57-61. Peng, S. K.; Tham, P.; Taylor, C. B.; Mikkelson, B. Am. J. Clin. Nutr. 1033-42. Seifter, J.; Baeder, D. H. Proc. Soc. Exptl. Biol. Med. 1956, 91, 42. Gray, M. F.; Laurie, T. D. V. Lipids 1971, 6, 836-43. Fieser, L. F. J. Am. Chem. Soc. 1953, 75, 5421. Peng, S-K.; Taylor, C. B.; Mosbach, Ε. H.; Huang, W. Y.; Hill, J . ; Mikkelson, B. Atherosclerosis 1982, 41, 395-402. Smith, L. L. "Cholesterol Autoxidation"; Plenum, New York. Simic, M. G.; Karel, M. "Autoxidation in Food and Biological Systems"; Plenum, New York. Roffo, A. H. Am. J . Cancer. 1933, 17, 42-57. Rosenheim, O.; Starling. W. W. Chem. & Ind. (London) 1933, 52, 1056 Windaus, Α.; Luders, Η. Z. Physiol. Chem. 1920, 109, 183. Haslewood, G. A. D. Biochem. J . 1941, 35, 708. Bergstrom, S.; Samuelson, B. Chp. 6, 233-48 (1961), "Autoxidation And Antioxidants Volume I." Ed. Lundberg, W.O.; Interscience Publishers, New York and London. Bischoff, F.; Rupp, J . J . Cancer Res. 1946, 6, 403-9. Fieser, L. F. Science 1954, 119, 710-6. Bergstrom, S.; Wintersteiner, O. J . Biol. Chem. (1941) 141, 597-610. Mosbach, E. H.; Neirenberg, M.; Kendall, F. E. (Abstract) Am. Chem. Soc. 10 C, Sept. (1952). Bischoff, F. J . Nat. Cancer Inst. (1957) 19, 977-8. Hieger, I. Brit. J. Cancer 1949, 3, 123-39. Hieger, I. Brit. Med. Bull. 1958, 14, 159-60. Bischoff, F.; Bryson, G. (Abst.) Am. Chem Soc. 62 C, Sept. (1960). Bischoff, F. Progr. Exp. Tumor Res. 1963, 3, 412-44. Bischoff, F. Adv. Lipid Res. 1969, 7, 165-244. Bischoff, F.; Bryson, G. Adv. Lipid Res. 1977, 15, 61-155. Dunn, T. B.; Heston, W. E.; Deringer, M. K. J. Nat. Cancer Inst. 1956, 17, 639-47.

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Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch005

RECEIVED July 11, 1983

6 Metabolism of Comutagens and Mutagens Produced from Tryptophan Pyrolysis PAUL P. LAU Department of Biochemistry and Molecular Biology, University of Texas Medical School at Houston, Houston, TX 77025 1

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch006

YUHSHI LUH Gulf Research and Development Company, Pittsburgh, PA 15230

Trp-P-2 and Norharman are the mutagen and comutagen shown to be metabolized by the reconstituted, purified rat-liver microsomal cytochrome P-448 system. The comutagenic action of Norharman upon aryl hydrocarbon hydroxylase activity is shown to be cytochrome P-448 dependent by immunochemical analysis. The hydroxylated products of Norharman and Harman may play an important role in their comutagenic action by fluidizing the microsomal or nuclear membranes. The enhancement or inhibitory effect of the comutagens on mutagenicity may be a net result of substrate inhibition and membrane fluidization in the microsomal or nuclear mixed-function-oxidase system. A schematic pathway is hypothesized for the chemical mutagenesis and comutagenesls of these tryptophan pyrolysis products. Two p o t e n t m u t a g e n i c p r i n c i p l e s , 3-amino-l,4-dimethvl-5Hp y r i d o ( 4 , 3 - b ) i n d o l e , Trp-P-1 and 3-amino-l-methyl-5H-pyrido(4,3-b) i n d o l e , T r p - P - 2 a n d two c o m u t a g e n s , Harman a n d Norharman were f o u n d i n t h e p y r o l y s i s o f t h e amino a c i d , D , L - t r * y p t o p h a n ( l ) . The same p y r o l y s i s p r o d u c t s were a l s o f o u n d i n c h a r r e d meat, smoke c o n d e n s a t e , c i g a r e t t e t a r a n d n a t u r a l l y o c c u r r i n g f o o d ( 2 - 4 ) . Numerous s t u d i e s o f t h e s e p y r o l y s i s p r o d u c t s o f t r y p t o p h a n a n d o t h e r amino a c i d s ( 1 ) have been r e p o r t e d b y S u g i m u r a a n d c o w o r k e r s (1,3). P y r o l y s a t e s o f t r y p t o p h a n , g l u t a m i c a c i d , l y s i n e , s e r i n e and o r n i t h i n e were shown t o have h i g h m u t a g e n i c i t y i n t h e Ames t e s t ( 5 ) . The chemical s t r u c t u r e s o f these mutagenic p r i n c i p l e s have been i d e n t i f i e d ( 8 ) a n d t h e y a r e a l l N - c o n t a 1 n i n g h e t e r o c y c l i c compounds o f t h r e e o r more r i n g s ( 8 ) , D u r i n g t h e i s o l a t i o n o f t h e s e p y r o l y s i s p r o d u c t s o f t h e amino a d d s and p r o t e i n s from t h e c h a r r e d p y r o l y s a t e s , Sugimura e t a l . Current address: Callery Chemical Company, Division of Mine Safety Appliances Co., Callery, PA 16024 1

©

0097-6156/83/0234-0099$06.00/0 1983 A m e r i c a n C h e m i c a l S o c i e t y

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch006

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FEEDS

(1,2) f o u n d t h a t t h e t o t a l m u t a g e n i c i t y d e c r e a s e d a f t e r t h e r e moval o f t h e f r a c t i o n s c o n t a i n i n g Harman and Norharman. Norharman and Harman have f o u n d t o enhance t h e m u t a g e n i c i t y o f T r p - P - 2 and T r p - P - 1 , B e n z o ( a ) p y r e n e and o t h e r mutagens. However, o t h e r w o r k e r s ( 9 , 1 0 ) f o u n d t h a t b o t h Norharman and Harman i n h i b i t e d t h e mutagene s i s o f T r p - P - 1 and T r p - P - 2 and c o v a l e n t DNA b i n d i n g o f B e n z o ( a ) p y r e n e i n v i t r o . L a t e r , Nagao e t al_. (5) r e p o r t e d t h a t t h e a c t i o n o f Norharman on c o m u t a g e n e s i s depended on t h e c h e m i c a l s t r u c t u r e o f t h e c h e m i c a l mutagens and t h e amount o f S-9 ( t h e c y t o s o l f r a c t i o n o f r a t - l i v e r microsomes p r e p a r a t i o n ) t h a t was added i n t h e Ames t e s t ( _ U , 1 2 ) . I t has been shown t h a t t h e e x p r e s s i o n o f m u t a g e n i c i t y and c o v a l e n t DNA b i n d i n g o f T r p - P - 1 and T r p - P - 2 r e q u i r e d m i c r o s o m a l a c t i v a t i o n i n the r e v e r t a n t mutation assay o r the forward mutation assay(12,19). N e b e r t e t a i . (14) showed t h a t t h e s e p y r o l y s i s p r o d u c t s f r o m amino a c i d s and p r o t e i n s r e q u i r e d t h e cytochrome P-450j w h i c h was d e f i n e d a s t h e groups o f t h e m u l t i p l e forms o f c y t o chrome P-450 i n d u c i b l e by p o l y c y c l i c a r o m a t i c compounds. The r o l e o f c y t o c h r o m e P-450 i n t h e m e t a b o l i c a c t i v a t i o n o f mutagens and c a r c i n o g e n s and t h e e x i s t e n c e o f t h e m u l t i p l e forms o f cytochrome P-450 i n h e p a t i c and e x t r a h e p a t i c t i s s u e s o f r a t s , r a b b i t s , human and v a r i o u s l i v i n g o r g a n i s m s i n c l u d i n g b a c t e r i a , y e a s t , and f r u i t f ! i e s ( 1 5 , 1 6 ) h a v e been r e v i e w e d . H a y a s h i e t a l ( 1 7 ) have shown t h a t Norharman and Harman bound t o DNA n o n c o v a l e n t l y by i n t e r c a l a t i o n c a u s i n g 17 u n w i n d i n g o f t h e s u p e r c o i l e d DNA d u p l e x . We have shown t h a t T r p - P - 1 and T r p - P - 2 bound t o DNA n o n c o v a l e n t l y ( 1 8 ) i n a d d i t i o n t o t h e i r c o v a l e n t DNA b i n d i n g . The n o n c o v a l e n t DNA b i n d i n g c o n s t a n t s were f o u n d t o c o r r e l a t e with the m u t a g e n i c i t y i n the forward mutation assay(19) when s i x o t h e r s y n t h e t i c a n a l o g s were s t u d i e d . The c o r r e l a t i o n o f n o n c o v a l e n t b i n d i n g t o DNA i n t h e absence o f m e t a b o l i c a c t i v a t i o n w i t h m u t a g e n i c i t y i n w h i c h m e t a b o l i c a c t i v a t i o n was r e q u i r e d , was c o n s i d e r e d as an unique c h a r a c t e r i s t i c o f these N - c o n t a i n i n g h e t e r o c y c l i c compounds. I t was a l s o s u g g e s t e d t h a t t h e mechanism o f m u t a g e n e s i s i n v o l v e s m e t a b o l i c a c t i v a t i o n f o l l o w e d by p h y s i o c h e m i c a l i n t e r a c t i o n w i t h DNA and f o r t h e s e compounds, t h e l a t t e r s t e p m i g h t be l i m i t i n o f o r t h e e x p r e s s i o n o f m u t a g e n i c i t y ( 1 9 ) . Umezewa e t al.(28) showed t h a t t h e r e was a c o m u t a g e n i c a c t i o n o f Norharman o n N - a c e t o x y - a m i n o f l u o r e n e ( A A F ) , w h i c h does n o t r e q u i r e m e t a b o l i c a c t i v a t i o n f o r c o v a l e n t DNA b i n d i n g . T h e r e f o r e t h e y p r o p o s e d t h a t t h e comutagens a l t e r e d t h e c o n f o r m a t i o n o f DNA w h i c h i n t u r n i n c r e a s e d t h e c o v a l e n t DNA b i n d i n g . T h i s meant t o be a n a l o g o u s t o t h e f i n d i n g o f Krugh and Young(21) t h a t Daunor u b i c i n and A d r i a m y c i n w h i c h i n t e r c a l a t e DNA c o v a l e n t l y , f a c i l i t a t e t h e b i n d i n g o f A c t i n o m y c i n D t o DNA. We, however, have shown t h a t t h e a l t e r a t i o n o f t h e DNA h e l i x by i n t e r c a l a t i o n o f Harman o r Norharman d i d n o t a f f e c t t h e a f f i n i t y o f T r p - P - 1 and T r p - P - 2 f o r DNA. The c o v a l e n t DNA b i n d i n g i n w h i c h m e t a b o l i c a c t i v a t i o n by cytochrome P-448 was r e q u i r e d , was shown t o be i n h i b i t e d by t h e a d d i t i o n o f Harman and N o r h a r m a n ( 1 8 ) .

6.

LAUANDLUH

Comutagens

and Mutagens

from

Tryptophan

101

The c o n f u s i o n o n t h e mechanism o f t h e c o m u t a g e n e s i s and muta­ genesis o f these p y r o l y s i s products, e s p e c i a l l y p e r t a i n i n g t o the enhancement and i n h i b i t i o n e f f e c t s o f Harman and N o r h a r m a n , c e n t e r s around the problem o f the lack o f c e r t a i n f i x e d v a r i a b l e s i n the experimentation, p a r t i c u l a r l y , the a v a i l a b i l i t y o f the p u r i f i e d enzymes i n v o l v e d i n t h e m e t a b o l i c a c t i v a t i o n , w h i c h c o n s t i t u t e t h e c y t o c h r o m e P-450 mixed f u n c t i o n o x i d a s e s y s t e m . We, t h e r e f o r e , u n d e r t a k e t h i s p r o b l e m t o e l u c i d a t e t h e mechanism o f m i c r o s o m a l m e t a b o l i s m o f t h e s e o y r o l y s i s p r o d u c t s w i t h t h e p u r i f i e d mixed f u n c t i o n oxidase(MFO) system.

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch006

M a t e r i a l s and

Methods

M a t e r i a l s : Harman and Norharman were p u r c h a s e d f r o m A l d r i c h Co. ( M i l w a u k e e , WIS). A l l c h e m i c a l s were r e a g e n t - g r a d e d . HPLC s o l v e n t s were HPLC g r a d e d . S y n t h e s i s o f C h e m i c a l Mutagens: A k i m o t o e t a]_(20) r e p o r t e d t h e f i r s t s y n t h e s e s o f p o t e n t mutagens, T r p - P - 1 and T r p - P - 2 i n 1977. The s y n t h e t i c schemes f o r T r p - P - 1 , [ 1 ] , a n d T r p - P - 2 , [ 2 ] were shown i n f i g u r e 1. 2 , 5 - D i m e t h y l - 4 - n i t r o p i c o l i n i c a c i d [ 4 ] was r e a d i l y ( 2 1 ) prepared from commercially a v a i l a b l e 2 , 5 - l u t i d i n e [ 3 ] . The r e ­ a c t i v e n i t r o group o f p i c o l i n i c a c i d [ 4 ] was s m o o t h l y s u b s t i t u t e d with O-phenylenediamine t o give m - a m i n o p i c o l i n i c a c i d [ 5 ] . Treat­ ment o f [ 5 ] w i t h n i t r o u s a c i d r e s u l t e d a b e n z o t r i a z o l y l p i c o l i n i c [ 6 ] i n q u a n t i t a t i v e y i e l d . T h e c a r b o x y l i c a c i d group on [ 6 ] was e a s i l y t r a n s f o r m e d t o an amino group v i a C u r t i u s r e a r r a n g e m e n t b y DPPA m e t h o d ( 2 2 ) , p r o d u c i n g [ 7 ] . Thermal d e c o m p o s i t i o n o f [ 7 ] t o e v o l v e n i t r o g e n i n t h e u s u a l w a y ( 2 3 ) y i e l d e d t h e d e s i r e d compound, 3 - a m i n o - l , 4 - d i m e t h y l - 5 H - p y r i d o ( 4 , 3 - b ) i n d o l e [ l ] ; m/e 211(M );vmax ( K B r ) : 1620,1600 c m " l ; δ ( C D 0 D ) : 3.32(3H,S), 2.73(3H,S), 7.13-7.33 (3H,m), 7.86(lH,m). The s y n t h e s i s o f T r p - P - 2 [ 2 ] s t a r t e d w i t h a n i n t e r m e d i a t e , i n d o l e - 2 - a c e t o n i t r i l e [ 9 ] ( 2 4 ) , prepared from commercially a v a i l a b l e indole-2-carboxylic acid[8j. Vilsmeier reaction(25) o f [9], with d i m e t h y l a c e t a m i d e a n d p h o s p h o r y l c h l o r i d e , gave 3 - a c e t y l i n d o l e - 2 a c e t o n i t r i l e [ 1 0 ] . C y c l i z a t i o n o f [10] on treatment w i t h m e t h a n o l i c ammonia and a r o m a t i z a t i o n i n v o l v i n g a h y d r o g e n t r a n s f e r o f t h e α-methylene group y i e l d e d the d e s i r e d 3-amino-l-methyl-5H-pyrido ( 4 , 3 b ) i n d o l e [ 2 ] ; m/e 197(M+); vmax(KBr) :1635,1605 c m " l ; δ (CD3OD): 2 . 3 ( 3 H , s ) , 5 . 9 2 ( l H , s ) , 6.53-7.00(3H,m), 7 . 3 5 ( l H , d ) . Takeda e t a l ( 2 6 ) p r e p a r e d mutagen T r p - P - 2 i n an a l t e r n a t i v e way as shown i n f i g u r e 2. I n d o l e - 2 - a c e t o n i t r i l e [ 9 ] p r e p a r e d f r o m t h e R e i s s e r t compound ( 2 7 ) . E t h y l 2 - c y a n o - l , 2 d i h y d r o q u i n o l i n e - l - c a r b o x y l a t e [ l l ] was c h o s e n a s an i n t e r m e d i a t e f o r t h e s y n t h e s i s o f T r p - P - 2 , T h e p h o t o p r o d u c t f r o m i r r a d i a t i o n o f [ 1 1 ] i n e t h e r a t 0-5OC i s t h e aliène [12]. Treatment o f [12] w i t h alumina o r s i l i c a gel gave[13]. This s t e p i s c o n s i d e r e d t o p r o c e e d by b a s e o r a c i d c a t a l y z e d i n t r a 3

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch006

X E N O B I O T I C S IN F O O D S A N D

Figure

1. Synthesis

of Trp-P-1

and Trp-P-2

by method

1.

FEEDS

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch006

LAU ANDL U H Comutagens and Mutagens from Tryptophan

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch006

104

X E N O B I O T I C S IN F O O D S A N D

FEEDS

m o l e c u l a r c y c l i z a t i o n ( 2 9 ) . Removal o f t h e N-l s u b s t i t u e n t i n I n d o l e - ! - c a r b o x y l a t e ( 1 3 j by m i l d h y d r o l y s i s w i t h p o t a s s i u m c a r ­ b o n a t e i n e t h a n o l a f f o r d e d 2-cyanomethyl i n d o l e [ 9 J . Subsequent Lewis a c i d ( A l C l 3 ) c a t a l y z e d c y c l i z a t i o n o f [ 9 ] i n t h e p r e s e n c e o f a c e t o n i t r i l e y i e l d e d T r p - P - 2 . The f o r m a t i o n o f [ 2 ] f r o m [ 9 ] i s proceeded by F r i e d e l - C r a f t s i n t e r m e d i a r y o f [14] f o l l o w e d by a r o m a t i z a t i o n v i a hydrogen m i g r a t i o n . The s u c c e s s f u l s y n t h e s i s o f T r p - P - 2 f r o m 2 - c y a n o m e t h y l i n d o l e [ 9 ] a l l o w e d us t o e n v i s a g e s i m i l a r s e q u e n c e s t o a f f o r d Trp-P-1 f r o m 2 - ( l - c y a n o ) e t h y l i n d o l e [ 1 7 ] . Due t o t h e n u c l e o p h i l i c p r o p e r ­ t i e s o f t h e i n d o l e r i n g a t C-3 p o s i t i o n , an e l e c t r o n - w i t h d r a w i n g group(eg. c a r b o x y l a t e ) i s r e q u i r e d t o e l i m i n a t e a l k y l a t i o n a t b o t h N-l and C-3 p o s i t i o n s i n f a v o r o f C-2atom t o t h e d e s i r e d c o n f i g u r a t i o n . The r e a d i l y a v a i l a b l e N - s u b s t i t u t e d 2 - c y a n o m e t h y l i n d o l e [ 1 3 ] was s e l e c t e d a s s t a r t i n g m a t e r i a l f o r o u r new p r o c e d u r e f o r t h e s y n t h e s i s o f Trp-P-1 a s shown i n F i g u r e 2. A k y l a t i o n o f [ 1 3 ] w i t h m e t h y l i o d i d e gave a m i x t u r e o f mono[15] and d i - a l k y l a t e d p r o d u c t s [ 1 6 ] w i t h sodium h y d r i d e a s t h e b a s e . By e m p l o y i n g l i t h i u m d i i s o p r o p y l i m i d e ( L D A ) i n d r y THF a t -78°C, a s o l e p r o d u c t o f e t h y l 2 - ( l - c y a n o ) e t h y l - i n d o l e - l - c a r b o x y l a t e [ 1 5 ] was o b t a i n e d . The s e l e c t i v e a l k y l a t i o n i n t h i s s t e p i s due t o t h e s t e r i c e f f e c t o f b u l k y LDA m o l e c u l e t o s u p p r e s s s e c o n d a l k y l a t i o n . M i l d h y d r o l y s i s o f [ 1 5 ] i n t h e same f a s h i o n w i t h d r y e t h a n o l i c p o t a s s i u m c a r b o n a t e gave [ 1 7 ] i n good y i e l d . P r e p a r a t i o n o f Enzymes: P r e p a r a t i o n o f r a t - l i v e r m i c r o s o m e s was p e r f o r m e d a s d e s c r i b e d ( 3 0 ) . P u r i f i c a t i o n o f c y t o c h r o m e P-450 and s e p a r a t i o n o f t h e m u l t i p l e forms were done a s d e s c r i b e d p r e v i o u s l y by Lau and S t r o b e l ( 3 0 ) . A n t i b o d y , IgG f r a c t i o n was p r e p a r e d a s d e s c r i b e d ( 3 0 ) . P-450 r e d u c t a s e was p r e p a r e d a c c o r d i n g t o t h e p r o c e d u r e o f Dignam and S t r o b e ! ( 3 1 ) . The r e c o n s t i t u t i o n o f t h e MFO s y s t e m w i t h t h e p u r i f i e d c y t o c h r o m e P-450, c y t o c h r o m e P-450 r e d u c t a s e and p h o s p h o l i p i d v e s i c l e s was p e r f o r m e d a c c o r d i n g t o Lu and C o o n ( 3 2 ) . C y t o c h r o m e P-448 was p r e p a r e d f r o m 3-MC m i c r o ­ somes o f m a t u r e S p r a g u e - D a w l e y r a t s . The h e p a t i c c y t o c h r o m e P-448 p r e p a r e d f r o m t h e s e m i c r o s o m e s i n d u c e d by 3 - m e t h y l c h o l a n t h r e n e a r e i n d e n t i c a ! i m m u n o l o g i c a l l y and s t r u c t u r a l l y t o t h o s e b y β N a p h t h o f l a v o n e a s shown by Lau e t a l ( 3 3 ) . T h e same f o r m o f c y t o ­ chrome P-448 has been p u r i f i e d f r o m o t h e r i n d u c e d m i c r o s o m e s s u c h as t h o s e b y PCB and TCDD(34). Microsomal Metabolism: A t y p i c a l r e a c t i o n o f microsomal metabol­ ism o r m e t a b o l i s m by t h e p u r i f i e d and r e c o n s t i t u t e d MFO s y s t e m c o n t a i n e d 1 ml o f aqueous s o l u t i o n : 0 . 0 7 5 v o l . o f d r u g i n m e t h a n o l , 0.1 mol p o t a s s i u m p h o s p h a t e ( p H 7 . 4 ) , lOymol M g C U , 0.75ymol NADPH and 0.15 v o l o f t h e enzyme s y s t e m . I n c u b a t i o n a t 37°C f o r t e n mi η was t e r m i n a t e d b y t h e a d d i t i o n o f 1 ml o f a c e t o n e on i c e and t h e m e t a b o l i t e s were e x t r a c t e d t w i c e w i t h e t h y l a c e t a t e . D r y MgSO^ was added and t h e l i q u i d p o r t i o n was d r i e d w i t h a s t r e a m o f n i t r o g e n g a s . A l l o f t h e s e r e a c t i o n s were c a r r i e d o u t under the yellow l i g h t .

6.

LAUANDLUH

Comutagens

and Mutagens

from

Tryptophan

105

When t h e a n t i b o d y t o c y t o c h r o m e P-450 was i n v o l v e d i n t h e e x p e r i m e n t , t h e P-450 IgG f r a c t i o n was added t o t h e c r u d e m i c r o somes b e f o r e t h e a d d i t i o n o f t h e d r u g a n d NADPH. The HPLC a n a l y s e s were p e r f o r m e d a s d e s c r i b e d . ( 3 5 )

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch006

Results and D i s c u s s i o n Microsomal M e t a b o l i s m o f T r p - P - 2 : T r p - P - 2 and AAF were m e t a b o l i z e d by t h e 3-MC r a t - l i v e r m i c r o s o m e s i n v i t r o . The e x t r a c t s w i t h b e n z e n e , d i e t h y l e t h e r , e t h y l a c e t a t e and e t h y l e n e c h l o r i d e were d r i e d b e f o r e i n j e c t i o n o n t o a r e v e r s e - p h a s e uBondapak C-18 c o l u m n . The HPLC p r o f i l e s o f t h e m e t a b o l i t e s o f T r p - P - 2 and AAF a r e v e r y s i m i l a r ( f i g u r e 3 ) . These m e t a b o l i t e s o f T r p - P - 2 have t h e same f l u o r e s c e n c e s p e c t r a w i t h e m i s s i o n maximum a t 332 nm. The UV s p e c t r a a r e i d e n t i c a l t o t h a t o f T r p - P - 2 , t h a t i s , w i t h a maximum a t 260 nm. The peak, which has t h e same r e t e n t i o n t i m e a s t h a t o f t h e N-OH p r o d u c t i n t h e AAF p r o f i l e , may be t h e N-OH m e t a b o l i t e o f T r p - P - 2 . D e t a i l e d c h e m i c a l c h a r a c t e r i z a t i o n o f e a c h peak i s u n d e r way. The s i m i l a r i t y o f t h e m e t a b o l i c p r o f i l e s o f t h e s e two c a r c i n o g e n s i s n o t u n e x p e c t e d a s t h e y have s i m i l a r c h e m i c a l s t r u c t u r e s . Cytochrome P-450s a r e known t o p e r f o r m N - h y d r o x y l a t i o n , a r e n e o x i d a t i o n a n d d e m e t h y l a t i o n ( 1 5 , 1 6 ) . These f u n c t i o n s s h o u l d not be i n t e r f e r e d w i t h by t h e i n t e r n a T " n i t r o g e n s i n t h e p o l y c y c l i c r i n g such a s t h o s e i n t h e T r p - P - 2 . It i s not s u r p r i s i n g t o see s i m i l a r m e t a b o l i c p r o f i l e s f o r T r p - P - 2 and AAF. Microsomal M e t a b o l i s m o f Norharman: Crude 3-MC microsomes m e t a b o l i z e d norharman i n v i t r o , a s shown i n F i g u r e 4. The m a j o r m e t a b o l i t e s o f norharman were e l u t e d a t e a r l i e r r e t e n t i o n t i m e s t h a n t h e p a r e n t compound. They c o u l d be some h y d r o x y l a t e d p r o d u c t s as t h e y showed more p o l a r p o t e n t i a l i n t h e r e v e r s e - p h a s e chromatography. The s i g n i f i c a n c e o f m i c r o s o m a l m e t a b o l i s m o f norharman i s b a s e d on our e a r l i e r h y p o t h e s i s t h a t norharman m i g h t u t i l i z e t h e same m i c r o s o m a l enzymes a s T r p - P - 2 ( 1 8 ) . Peura e t al_(36) h a s shown an i n c r e a s e d membrane f l u i d i t y c a u s e d by tetraïïydro-3carbolines(tetrahydroharman) in a recent p u b l i c a t i o n . This suggests t h a t norharman, a f t e r i t i s h y d r o x y l a t e d , a c t s as a f l u i d i z i n g a g e n t f o r t h e m i c r o s o m a l membrane which i n t u r n c o u l d i n c r e a s e t h e a c t i v i t y o f c y t o c h r o m e P-450 and t h e membrane embedded c y t o c h r o m e P - 4 5 0 - r e d u c t a s e . The change o f a c t i v i t y due t o f l u i d i z a t i o n o f t h e m i c r o s o m a l membrane may be d e p e n d e n t upon t h e c o n c e n t r a t i o n o f t h e m i c r o s o m a l p r o t e i n s a s w e l l a s t h e amount o f t h e comutagens and mutagens a d d e d . Whether i t r e s u l t s i n a t o t a l n e t i n c r e a s e o r d e c r e a s e may w e l l depend on t h e b a l a n c i n g o f t h e e f f e c t o f s u b s t r a t e i n h i b i t i o n and t h a t o f enhancement d u e t o f l u i d i z a t i o n o f t h e m i c r o s o m a l membrane.

XENOBIOTICS

IN F O O D S A N D

FEEDS

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106

Figure 3. Microsomal metabolism of Trp-P-2 and AAF. HPLC profiles of organic solvent extracts of the metabolized mutagens. Rat livers (3-MC microsomes) were used. HPLC was performed with a stainless steel reverse phase C18 column.

Figure 4. Microsomal metabolism of norharman. HPLC profile of ethyl acetate extract. A similar reaction was performed for control, in which microsomes were not added and there were no metabolites eluted. HPLC was performed with a C-18 radial compression module.

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Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch006

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C o m u t a g e n i c A c t i o n o f Harman and Norharman: When t h e r e c o n s t i ­ t u t e d MFO s y s t e m , w h i c h c o n t a i n e d 3-MC i n d u c e d c y t o c h r o m e P-448, was u s e d t o s t u d y t h e e f f e c t s o f Norharman and Harman on t h e h y d r o x y l a t i o n o f B e n z o ( a ) p y r e n e , t h e y b o t h showed an enhancement e f f e c t u n d e r t h e o p t i m a l e n z y m a t i c c o n d i t i o n s . Norharman, however, showed a l a r g e r a c t i v i t y i n c r e a s e i n B e n z o ( a ) p y r e n e h y d r o x y l a t i o n than Harman(Figure 5 ) . I t a s c e r t a i n s t h a t the i n h i b i t o r y e f f e c t s o f t h e s e c a r b o l i n e s o n m u t a g e n i c i t y a r e n o t d e p e n d e n t on c y t o ­ chrome P-448 i n t h e s e n s e o f s i m p l e s u b s t r a t e i n h i b i t i o n . One may a r g u e t h a t t h e r e c o u l d be some c o n t r i b u t i o n o f t h e c o m u t a g e n i c a c t i v i t y p l a y e d b y t h e o t h e r m u l t i p l e forms o f c y t o ­ chrome P-450 t h a n t h a t o f c y t o c h r o m e P-448 i n t h e 3-MC m i c r o s o m e s . I t i s t r u e t h a t t h e r e a r e two o t h e r m a j o r f o r m s o f c y t o c h r o m e P-450 have been p u r i f i e d f r o m 3-MC i n d u c e d r a t - l i v e r m i c r o s o m e s (34) a n d f o u r o t h e r m a j o r forms o t h e r t h a n c y t o c h r o m e P-448 have been p u r i f i e d f r o m 6 N a D h t h o f l a v o n e r a t microsomes{30). A l t h o u g h t h e c o m u t a g e n i c a c t i o n s o f t h e s e compounds have n o t been s t u d i e d w i t h t h e s e p u r i f i e d forms o f c y t o c h r o m e P-450, t h e a v a i l a b i l i t y o f t h e a n t i b o d y IgG f r a c t i o n t o t h e p u r i f i e d c y t o c h r o m e P-448 a l l o w e d us t o s t u d y t h i s mechanism o n t h e m i c r o s o m a l l e v e l . The HPLC p r o f i l e s i n F i g u r e 6 shows a n o v e r a l l d e c r e a s e i n B e n z o ( a ) p y r e n e m e t a b o l i t e f o r m a t i o n upon t h e a d d i t i o n o f IgG P-448 f r a c t i o n . I n t e r e s t i n g l y t h e peak c o r r e s p o n d i n g t o t h e 3 , 6 - d i o n e decreases i n r e l a t i v e percentage o f a l l the ethyl acetate extract a b l e m e t a b o l i t e s . When t h e r e c o n s t i t u t e d p u r i f i e d c y t o c h r o m e P-448 s y s t e m was u s e d , t h e p r e s e n c e o f Norharman e n h a n c e d t h e f o r m a t i o n o f 3 , 6 - d i o n e ( F i g u r e 7 ) . T h e r e s u l t s shown h e r e s t r o n g l y s u g g e s t t h a t t h e c o m u t a g e n i c a c t i o n o f Norharman i s c y t o c h r o m e P-448 d e p e n d e n t . Conclusion T h e r e a r e s e v e r a l p o s t u l a t e s t h a t can be made b a s e d o n t h e d a t a shown i n t h i s work and t h o s e p u b l i s h e d e a r l i e r . T r a n s l o c a t i o n o f Mutagens and Comutagens a c r o s s C e l l u l a r membranes Is Enhanced by β C a r b o l i n e M e t a b o l i t e s " : y - C a r b o l i n e s ( T r p - P - l a n d T r p - P - 2 ) and β - c a r b o l i n e s ( H a r m a n and Norharman) a r e r e a d i l y w a t e r s o l u b l e and hence i s i n s o l u b l e , r e l a t i v e l y , i n h y d r o p h o b i c membranes. The β - c a r b o l i n e s a f t e r t h e y a r e h y d r o x y l a t e d c a n f l u i d i z e membranes t h e r e f o r e i t r e n d e r s t h e h y d r o p h o b i c p o l y c y c l i c s u c h a s B e n z o ( a ) p y r e n e more a c c e s s i b l e t o t h e membrane embedded MFO s y s t e m . In mammalian c e l l s , t h i s can be a two s t e p mechanism: The MFO i n t h e e n d o p l a s m i c r e t i c u l u m may f i r s t h y d r o ­ xy l a t e t h e comutagens a n d t h e h y d r o x y l a t e d comutagens t h e n f l u i d ­ i z e t h e n u c l e a r membrane e n a b l i n g t h e h y d r o p h o b i c p o l y c y c l i c s t o be i n c o n t a c t w i t h t h e MFO s y s t e m w h i c h i s embedded i n t h e n u c l e a r e n v e l o p e ( 3 7 ) . T h e a c t i v a t i o n o f mutagens by n u c l e a r c y t o c h r o m e P-450 w o u l d c a u s e t h e s u b s e q u e n t DNA b i n d i n g w h i c h t h e n t r i g g e r s the i n i t i a t i o n o f mutagenesis.

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch006

6.

LAUANDLUH

Comutagens

and Mutagens

from

Tryptophan

109

Norharman Added J Ο

S

•Η

fin

-Ρ

Ο

cd Η on ο Η

Harman A d d e d

5

10 Time o f

15 Incubation

20 (min)

Figure 5. Metabolism of comutagens by cytochrome P-448. The purified enzymes used were the reconstituted cytochrome P-448 system. The relative increase was computed by substrating the ethyl acetate extractable radioactivity in counts per minute from that of a corresponding control experiment in which BP was metab­ olized in the absence of comutagens.

110

IN F O O D S A N D F E E D S

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XENOBIOTICS

Figure 6. Microsomal metabolism mutagens. HPLC was performed

of aryl hydrocarbon in the presence of cowith two connected stainless steel C-18 columns.

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch006

LAU ANDL U H

Comutagens

and Mutagens

from

Tryptophan

Figure 7. Metabolism of aryl hydrocarbon by the purified cytochrome P-448 the presence of norharman. HPLC was performed as described in Figure 6.

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The Enhancement o r t h e I n h i b i t o r y E f f e c t o f t h e Β - C a r b o l i n e s I s a Net R e s u l t o f S u b s t r a t e I n h i b i t i o n and Membrane F l u i d i z a t i o n : We showed e a r l i e r ( 1 8 ) t h a t t h e p r e s e n c e o f Harman o r Norharman d i d n o t i n t e r f e r e t h e r e v e r s i b l e n o n c o v a l e n t DNA b i n d i n g f o r T r p - P - 2 , b u t i n h i b i t e d i r r e v e r s i b l e c o v a l e n t DNA b i n d i n g i n v i t r o upon t h e m e t a b o l i c a c t i v a t i o n b y t h e c r u d e m i c r o s o m a l f r a c t i o n i s o l a t e d from 3-MC p r e t r e a t e d r a t l i v e r s . Drug i n t e r c a l a t i o n s t a b i l i z e s t h e DNA d u p l e x . The u n s t a b l e A-T r i c h r e g i o n s i n a DNA d u p l e x n o r m a l l y can h a v e t r a n s i e n t l y d e n a t u r e d s t r u c t u r e i n a n o t h e r w i s e f u l l y d u p l e x i n aqueous s o l u t ­ ion o f p h y s i c o l o g i c a l c o n d i t i o n s , Ethidium bromide, a c l a s s i c a l i n t e r c a l a t o r and a p o s i t i v e l y c h a r g e d m o l e c u l e o f p l a n a r s t r u c t u r e can e v e n r e n a t u r e f u l l y d e n a t u r e d c l o s e d c i r c u l a r DNA(38), T h e i n h i b i t o r y e f f e c t o f E t h i d i u m b r o m i d e upon s i n g l e s t r a n d e d DNA n u c l e a s e i s a n o t h e r e v i d e n c e f o r t h e s t a b i l i z t i o n o f DNA d u p l e x by t h e i n t e r c a l a t i v e d r u g ( 3 9 ) . We must r e c o g n i z e t h a t Norharman and T r p - P - 2 b e i n g p o s i t i v e l y c h a r g e d i n t e r c a l a t o r s a l s o can s t a b i l i z e t h e DNA d u p l e x . I f Norharman d e s t a b i l i z e d t h e DNA d u p l e x , t h e comutagens w o u l d n o t have unwound t h e s u p e r c o i l e d DNA, I n f a c t , Norharman was shown t o unwind s u p e r c o i l e d DNA(17). T h e r e f o r e , we c a n n o t e n v i s i o n t h a t t h e comutagen b i n d i n g t o DNA w o u l d i n any way f a c i l i t a t e t h e DNA b i n d i n g o f t h e mutagens e i t h e r i n t h e c o v a l e n t o r n o n c o v a l e n t mode. The a l t e r n a t i v e e x p l a n a t i o n must I n v o l v e t h e MFO s y s t e m . T h e p o s s i b l e mechanism f o r t h e i n h i b i t o r y e f f e c t o r enhancement e f f e c t o f Norharman upon c o v a l e n t DNA b i n d i n g and m u t a g e n i c i t y must be t h e r e s u l t s o f t h e n e t b a l a n c i n g o f s u b s t r a t e I n h i b i t i o n and membrane f l u i d i z a t i o n o f t h e m i c r o s o m a l membrane o r o f t h e l i p i d v e s i c l e s i n t h e r e c o n s t i t u t e d MFO s y s t e m . A s c h e m a t i c pathway o f the metabolism o f these tryptophan p y r o l y s i s products i s p o s t u l a t e d a s shown i n F i g u r e 8. P l a u s i b l e Mechanism f o r A d d u c t F o r m a t i o n o f T r p - P - 2 and DNA: H a s h i m o t o e £ a l ( 4 0 ) have i d e n t i f i e d one o f t h e m a j o r DNA a d d u c t [ 2 ] f o r T r p - P ^ 2 l T J ( F i g u r e 9 ) . The c o v a l e n t b i n d i n g s i t e on t h e g u a n i n e i s a t t h e C-8 p o s i t i o n , The e x o c y c l i c amino g r o u p i n p y r i d o ( 4 , 3 b ) i n d o l e s i s a p r e r e q u i s i t e f o r e x p r e s s i o n o f mutaaeni c i t y ( 1 9 ) . Since the s t r u c t u r a l l y r e l a t e d 2-aminofluorene(AF)[3] exerts mutagenicity through the a c t i v a t e d s p e c i e s N-hydroxy-AF[4], i t seems t h a t t h e m u t a g e n i c p y r i d o ( 4 , 3 - b ) i n d o l e s may be a c t i v a t e d i n t h e same f a s h i o n s i n c e t h e e x o c y c l i c amino g r o u p i s r e q u i r e d i n t h e e x p r e s s i o n o f t h e m u t a g e n i c i t y f o r T r p - P - 2 , A s we have shown t h a t a peak o f s i m i l a r r e t e n t i o n a s t h a t o f t h e N-OH A F was o b s e r v e d i n t h e T r p - P - 2 p r o f i l e and, t h e w e l l known f u n c t i o n o f N - d e m e t h y l a t i o n b y t h e h y d r o x y l a t i o n o f t h e c y t o c h r o m e P-450 s y s t e m , we t h u s p r o p o s e t h e f o l l o w i n g mechanism f o r t h e C-8 g u a n i n e a d d u c t formation: It i s q u i t e p l a u s i b l e t h a t the m e t a b o l i c a l l y a c t i v a t e d Trp-P-2 is the corresponding hydroxylamine [5] o r i t s e s t e r s [ 6 ] , The e l e c t r o n - d e f i c i e n c y n a t u r e o f i m i n o g r o u p i n g u a n i n e m o l e c u l e may

Figure

frD-P-2(0H)n

frp-P-2(-N0H)

Reversible Non-covalent Binding

pathway for the chemical mutagenesis phan pyrolysis products.

of the

trypto­

Carcinogenesis

Tumorogenesis

I

ι

Initiation of Mutagenesi s

Irreversible Covalent TrD-P-2-DNA Adducts

and comutagenesis

Mixed-Function CO c Oxidase System .··" (-) Reconstituted ONA ° Cyt. P-448 or Crude Microsomes NADPH; "Membrane bindin< norharman (OH) η

NADPH

Reversible Non-covalent Binding

Enhancement due to membrane fluidization Inhibition due to substrate inhibition No net effect due to reversibility

8. A schematic

(+) (-) (+)

(+)

Comutagens:Norharman Harman

TRYPTOPHANE ο

Mutagens: Trp-P-1 Trp-P-2

(+)

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Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch006

114 X E N O B I O T I C S IN F O O D S A N D FEEDS

6.

L A UA N DL U H

Comutagens

and Mutagens

from

Tryptophan

115

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch006

c r e a t e t h e n u c l e o p h i l i c i t y o f t h e C-8 atom i n t h a t m o l e c u l e t h r o u g h e l e c t r o n - p r o t o n s h i f t . N u c l e o p h i l i c d i s p l a c e m e n t o f good l e a v i n g groups such a s , c a r b o x y l a t e o r s u l f a t e e s t e r s , i n t h e a c t i v a t e d T r p - P - 2 by g a a n i n e a t t h e C-8 p o s i t i o n , f o l l o w e d by a p r o t o n m i g r a t i o n , would g i v e t h e i d e n t i f i e d adduct [ 2 ] .

Literature Cited 1. Kosuge, T . , Tsuji, K., Wakabayashi, K., Okamoto, T . , Shudo,K., Titaka, Y . , Itai, Α . , Sugimura, T . , Kawachi, T., Nagao,M., Yahagi, T., and Seino, Y. Chem. Pharm. Bull. 1978, 26, 611-9. 2. Sugimura, T., Kosuge, T . , Tsuji, Κ., Wakabayashi, K., Iitaka, Y., and Itai, A. Proc. Japan Acad. Sci.1977,53,58. 3. Sugimura, T . , Sato, S., Nagao, Μ., Yahagi, T . , Matsushuima,T., Seino, Y . , Takeuchim T."Fundamentals in Cancer Prevention"; University of Tokyo Press, Tokyo, 1976;191-215. 4. Poindexter, Ε. H. and Carpenter, R.D.Phytochemistry,1962,1, 215-221. 5. Nagao, M., Honda, Μ.,Seino,Y., Yahagi,T. and Sugimura, T. Cancer Letters, 1977,2,221-226. 6. Matsumoto, T . , Yoshida, D., Mizusaki, S. and Okamoto H. Mutat. Res. 1977,481,279-286. 7. Akimoto, H . , Kawai, Α . , Nomura, Η.,Nagao, Μ., Kawachi, T . , and Sugimura, T. Chem. Lett. 1977, 1061-4. 8. Sugimura, T . , Nagao,M., Kawachi, T., Honda, M., Yahagi, T., Seino, Y . , Sato, S. and Matsukura, N. in "Origins of Human Cancer", Hiatt, H.H., Watson, J.D. and Winsten, J.A.(Cold Spring Harbor Laboratory, Cold Spring Harbor, NY), Eds., 1977,1561-1577. 9. Levitt, R.C., Legraverend, C . , Nebert, D. W., and Pelkonen, O. Biochem. Biophys. Res. Comm. 1977, 79, 1167-75. 10. Chang, C . , Castellazzi,M., Glover, T.W. and Trosko, J . E. Cancer Res. 1978, 38, 4527-33. 11. Ames, B.N. Sims, P. and Grover, P.L. Science, 1972,176,47-9. 12. Ames, B.N., Gurney, E. G . , Miller, J. A. and Bartsch, H. Proc. Natl. Acad. Sci. USA 1972, 69, 3128-32. 13. Skopek, T. R., Liber, H. L., Krolewski, J . J. and Thilly, W. G. Proc. Natl. Acad. Sci. USA 1978, 75, 410-4. 15. Coon, M . J . , Vermilion, J. L., Vatsis, K. P., French, J . S . , Dean, W. L.,and Haugen, D. A."Concepts in Drug Metabolism" Ed., Jerina, D.M.ACS Series, Washington D. C . , 1977; p.46-71. 16. Coon, M.J., and Vatsis, K.P."Polycyclic Hydrocarbons and Cancer"Gelboin, H.V., and Tso, P.O.P., Eds; Academic Press: New York,1978,, Vol. I, p.335-360. 17. Hayashi, Κ., Nagao, M. and Sugimura, T. Nucl. Acid. Res.1977, 4, 3679-85. 18. Lau, P. P. and Luh, Y. Biochem. Biophy. Res. Comm. 1979,89(1), 188-194. 19. Pezzuto,J.M., Lau, P. P., Luh, Y . , Moore,P.D;Wogan,G.N.and Hecht,S.Proc.Natl.Acad.Sci.USA.1980,77,1427-31.

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20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40.

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Akimoto,H., Kawai, A. and Nomura, H. Chem. Lett. 1977,1061-4. Matsumura, E., Ariza,M. and Ohifuji,Τ.Βull. Chem. Soc.Japan, 1970,43,3210. Ninomiya, K., Shioiri, T., and Yamada, S.Chem. Pharm. Bull. (Tokyo) 1974, 22, 1398. Robinson, R.and Thornley, J . Chem. Soc. 1925, 125,2169. Schindler, W. Helv. Chim. Acta.1957, 40, 2156. Antony, W. C. J. Org. Chem. 1960, 25, 2409. Takeda, K., Ohta, T . , Shudo, Κ., Okamoto,T., Tsuji, K. and Kosuge, T. Chem. Pharm. Bull. Japan, 1977, 25(6), 2145-6. Ikeda, M., Matsugashita, S. and Tamura, Y . , J. Chem. Soc. Perkin Trans I, 1976,2587-90. Umezewa, K., Shirai, Α . , Matsushima, T . , and Sugimura, T. Proc. Natl. Acad. Sci. USA. 1978, 75, 928-30. Caserio, M.C."Selective Organic Transformations" Thygarayan, Wiley,Eds; London, Vol I, p.272. Lau, P. P. and Strobel, H. W. J. Biol. Chemistry,1982,257(9), 5257-62. Dignam, J . D., and Strobel, H.W. Biochemistry, 1977,16,111623. Lu, Α.Y.H., Strobel, H.,and Coon, M.J. Mol. Pharmacol.1970, 6, 213-220. Lau, P. P., Pickett, C.B., Lu, Α.Y.H. and Strobel, H.W. Arch. Biochem. Biophys. 1982, 218(2), 472-7. Ryan, D. E., Thomas, P. E., Korzeniowski, D. and Levin, W. J. Biol. Chem. 1979, 254, 1365-74. Holder, G., Yagi, H . , Dansette, P., Jerina, D. M., Levin, W., Lu, Α.Y.H. and Conney, A.H. Proc. Natl. Acad. Sci. USA 1974 71(11), 4356-60. Peura, P., Mackenzie, P., Koivusaari, U.and Lang, M. Mol. Pharm. 1982, 22, 721-24. Bresnick, E., Vaught, J . B., Chuang, A.H.L., Stoming, T.A. Bockman, D. and Mukhtar, H. Arch. Biochem. Biophys. 1977,181, 257-69. Lau, P. P. and Gray, H. B. Nucl. Acid. Res. 1980, 8, 673-701. Lau, P. P. and Gray, H. B. Nucl. Acid. Res. 1979, 6, 331-57. Hashimoto, Y . , Shudo, Κ., and Okamoto, T. Chem. Pharm. Bull. 1979, 27(4), 1058-60.

RECEIVED June

3, 1983

7 Mutagen Formation in Processed Foods 1

C. A. KRONE and W. T. IWAOKA

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch007

Institute for Food Science and Technology, University of Washington (WH-10), Seattle, WA 98195 The role of cooking in the formation of mutagenic substances in food is well established. Broiling, grilling and frying of foods, especially those high in protein, can produce substances which cause mutation in bacterial and mammalian in vitro systems. A large class of foods which also undergoes heat treatment but which has been overlooked in mutagen studies is commercially processed foods. This paper describes a survey of mutagenicity in various commercially heated foods, investigates the process of mutagen formation in two canned products and discusses the implications for the food industry and regulatory agencies if the mutagens formed during processing are eventually found to be carcinogenic. Many carcinogens and mutagens have been detected i n cooked foods. These compounds may be present as a r e s u l t of smoke condensation on the product during heat treatment or of thermal degradation of food components. The c r u s t of c h a r c o a l - b r o i l e d beefsteak can contain between 8 and 50 yg benzo(a)pyrene/kg (1). Wood smoked meats and f i s h c o n t a i n a v a r i e t y of p o l y c y c l i c aromatic hydro­ carbons (2). F i s h and beef g r i l l e d over a gas flame c o n t a i n substances which are mutagenic i n the Ames Salmonella assay (3). Some of these mutagens have been i d e n t i f i e d as p y r o l y s i s products of the amino a c i d s tryptophan and glutamic a c i d (4*5). The potent mutagens 2-amino-3 methylimidazo [4,5-f] q u i n o l i n e (IQ), 2-amino-3,4 - dimethylimidaζο [4,5-f] q u i n o l i n e (MelQ) and 2-amino3, 8-dimethylimidaζο [4,5-f] q u i n o x a l i n e (Me-IQ ) were a l s o i s o l a t e d from b r o i l e d f i s h and beef 06,7) . The above heat t r e a t ­ ments used temperatures >300°C; however, mutagenic substances can a l s o form when foods are cooked at the lower temperatures more commonly employed i n North America. F e l t o n e t a l . ( 8 ) have shown X

1

Current address: Department of Biochemistry and Biophysics, University of Hawaii at Manoa, Honolulu, HI 96822 0097-6156/83/0234-0117$06.00/0 © 1983 A m e r i c a n C h e m i c a l S o c i e t y

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that mutagens are formed i n a wide v a r i e t y of beef, pork, p o u l t r y , f i s h and egg products when they are b r o i l e d or f r i e d at 200-300°C. Both the length of cooking time and temperature have been shown to be important f a c t o r s i n mutagen formation during cooking. P a r i z a et a l . (9) found that ground beef f r i e d at 143°C contained few mutagenic substances even a f t e r cooking f o r 20 min to a w e l l done s t a t e . However, f r y i n g at 190°C f o r l e s s than 10 min l e d to s i g n i f i c a n t q u a n t i t i e s of mutagens. Krone and Iwaoka (10) showed that the mutagenicity of f i s h f r i e d a t 190°C increased r a p i d l y a f t e r a 6 min l a g p e r i o d . In general, longer cooking times and higher temperatures i n c r e a s e the extent of mutagen formation during cooking. The M a i l l a r d browning r e a c t i o n i s a l s o o f t e n i m p l i c a t e d i n the development of mutagens i n heated foods. Dichloromethane e x t r a c t s of browning r e a c t i o n model systems (heated sugar p l u s amine mixtures) are mutagenic i n the Ames t e s t u s i n g JS. typhimurium s t r a i n s TA98 and TA100 with and without microsomal a c t i v a t i o n (11,12). Substances which induced chromosome a b e r r a t i o n s i n Chinese hamster ovary (CHO) c e l l s and m i t o t i c recombinations and mutation i n Saccharomyces c e r e v i s i a e s t r a i n D5 are a l s o found i n these browning model systems (13). Caramelized sugars and commercial caramel powder (used as a c o l o r a n t i n beverages, beer, gravy mixes, soups, etc.) possess chromosome damaging a c t i v i t y i n CHO c e l l s (14). Mutagens were reported to be present i n heated high s t a r c h content foods (potatoes, pancakes, b i s c u i t s , toasted bread) and were a t t r i b u t e d to browning r e a c t i o n s (15). These mutagens may, however, have been a r t i f a c t s , caused by the use of ammonium ions ((NH4)2S04 and NH4OH) i n the mutagen e x t r a c t i o n procedure. I t has been shown by Iwaoka et a l (16) that when sodium analogs of the ammonium compounds (Na2S04 vs. (NH^^SO^ and NaOH vs. NH^OH) were used, no mutagens were detected i n a commercial b i s c u i t product. S i g n i f i c a n t q u a n t i t i e s of mutagens were produced when ammonium ions were present during the e x t r a c t i o n of the same b i s c u i t product. In a l i m i t e d survey of foods which had undergone M a i l l a r d browning ( c o r n f l a k e s , r i c e c e r e a l , c r a c k e r s , coconut cookies, bread crumbs), P a r i z a et a l (17) detected low l e v e l s of mutagenicity of most products. Nearly a l l of the above s t u d i e s involved h e a t i n g processes which take p l a c e i n the home, while foods heat processed at the commercial l e v e l have been s u b s t a n t i a l l y overlooked. Battered and breaded products (which are p a r t i a l l y cooked before f r e e z i n g and d i s t r i b u t i o n ) undergo M a i l l a r d browning which, as has been mentioned above, can produce mutagenic substances. Canning of food products involves r e l a t i v e l y low temperatures compared to f r y i n g (-115°C vs. ~200°C) but r e q u i r e s extended p r o c e s s i n g times of greater than one and one h a l f hours. Considering the i n f l u e n c e of both time and temperature, the presence of mutagenically a c t i v e substances i n canned foods seemed probable. T h i s paper r e p o r t s on the mutagen content of a v a r i e t y of commercially heated foods and i n v e s t i g a t e s the process of mutagen formation i n two canned products.

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Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch007

M a t e r i a l s and Methods Food Samples. Commercially processed f r o z e n and canned products were purchased a t l o c a l markets. The f r o z e n products were subjected to the aqueous e x t r a c t i o n procedure described below, without f u r t h e r heating ( d i r e c t l y from the package) or a f t e r being heated by the method recommended by the manufacturer. For the canned products, the e n t i r e contents of each can was e x t r a c t e d using the methanol procedure. In the s t u d i e s on the process of mutagen formation during canning, two products were used, ground beef and pink salmon. These products were placed i n 307 χ 200.25 s i z e cans, sealed with a vacuum s e a l e r and r e t o r t e d f o r 85 min a t 117°C. P r i o r to canning some p o r t i o n s of ground beef were a l s o dispersed i n an equal weight of water, the mixture heated to 100°C f o r 10 min and the s o l i d s separated from the stock by f i l t e r i n g through g l a s s wool. The f i l t e r e d s o l i d s and stock were placed i n separate cans, sealed and heat processed as above. Several precanning treatments were a l s o performed on f r e s h pink salmon. The f i s h were cut i n t o steaks (~2 cm t h i c k ) and one p o r t i o n f r o z e n overnight at -18°C. Fresh and thawed f r o z e n steaks were wrapped i n f o i l and steamed (100°C) f o r 1 hr. The f l u i d which l e f t the f i s h t i s s u e during steaming (stock) was drained from the f l e s h , and the stock and remaining f l e s h were packed i n separate cans and processed. Aqueous E x t r a c t i o n Procedure. The f r o z e n products (mostly battered and breaded seafoods) were homogenized with four volumes (w/v) of d i s t i l l e d water, saturated with Na2S0^ and adjusted to pH 2.5 with HC1. When the p r o t e i n s had p r e c i p i t a t e d the homogenate was f i l t e r e d through g l a s s wool and Whatman No. 1 f i l t e r paper, u s i n g s l i g h t s u c t i o n . The f i l t r a t e was e x t r a c t e d three times with 20 ml g l a s s d i s t i l l e d dichloromethane (CH2CI2) per 100 ml aqueous to give the a c i d i c organic e x t r a c t . The aqueous phase was adjusted to pH 10 with 50% NaOH and again p a r t i t i o n e d three times with CH 2 Cl2 (basic organic e x t r a c t ) . The organic e x t r a c t s were d r i e d over Na2S04 and evaporated to dryness i n a r o t a r y evaporator. The residues were d i s s o l v e d i n a known volume of d i m e t h y l s u l f o x i d e (DMS0) and a l i q u o t s tested i n the Ames assay. Methanol E x t r a c t i o n Procedure. The canned foods were homogenized with three volumes (w/v) of methanol, the homogenate f i l t e r e d through Whatman No. 1 f i l t e r paper o v e r l a i d with g l a s s wool. The methanol was removed from the f i l t r a t e by r o t a r y evaporation and the residue that remained was d i s s o l v e d i n a volume of d i s t i l l e d water (w/v) equal to four times the o r i g i n a l weight of food product. T h i s aqueous mixture was adjusted to pH 2.5 and p a r t i t i o n e d three times with CH2CI2 as i n the above procedure. The aqueous phase was made b a s i c (pH 10) and e x t r a c t e d three times.

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Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch007

The CH2CI2 e x t r a c t s were d r i e d , evaporated and d i s s o l v e d i n DMSO as described p r e v i o u s l y . Mutagenicity T e s t i n g . A l i q u o t s of each e x t r a c t equivalent to 80 g of o r i g i n a l food product (80 g Eq) were tested f o r mutagenicity according to the procedure described by Ames e t a l (18) using Salmonella typhimurium s t r a i n 1538, TA98, and TA100. E x t r a c t s were t e s t e d with and without the a d d i t i o n of 80 μΐ Arochlor induced r a t l i v e r microsome preparation (S9) per p l a t e . Results are reported e i t h e r as mean r e v e r t a n t s produced ± standard d e v i a t i o n (the mean number of spontaneous r e v e r t a n t s have been subtracted from the t o t a l c o l o n i e s on each p l a t e ) o r as a mutagenic a c t i v i t y r a t i o (MAR) which equals the number of r e v e r t a n t s on p l a t e s with food e x t r a c t s d i v i d e d by the number of spontaneous r e v e r t a n t s . Results and D i s c u s s i o n In a t y p i c a l commercial f i s h s t i c k operation, f i s h s t i c k s or p o r t i o n s are cut from frozen blocks of f l e s h , the pieces are breaded and b a t t e r e d , cooked i n vegetable o i l (~200°C), packaged and r e f r o z e n . The degree of browning that occurs during cooking v a r i e s with time o f cooking, type of breading, e t c . Small p i e c e s of breading may be detached during the cooking process, darken r a p i d l y , sometimes adhering to other pieces of product and p o s s i b l y c o n t r i b u t i n g to mutagen content. The mutagenic a c t i v i t y of the a c i d i c and b a s i c e x t r a c t s o f a v a r i e t y of battered and breaded seafood products i s shown i n Table I .

Table I . Mutagenicity

of A c i d i c and Basic E x t r a c t s of Seafood Products MAR* (80 g Eq) Acidic Extract Basic E x t r a c t

Product Breaded f i s h f i l l e t s Breaded clams Breaded shrimp French f r i e d f i s h s t i c k s Whitemeat f i s h i n b a t t e r Minced shrimp s t i c k s

1.6 1.2 0.9 2.2 0.7 1.9

1.8 1.3 1.1 1.8 0.4 1.0

*w . .„ . .. .. No. of r e v e r t a n t s on p l a t e s with Mutagenic A c t i v i t y Ratio = extrac No. oft spontaneous r e v e r t a n t s D

r

S_. typhimurium s t r a i n 1538 with 80 μΐ S9 (mean spontaneous = 24 r e v e r t a n t s )

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Using the c r i t e r i o n that an MAR of 2.5 i n d i c a t e s a p o s i t i v e t e s t , none of the products assayed appeared to c o n t a i n mutagenic substances. In general, when these products were heated both according to manufacturers' i n s t r u c t i o n s and to an overcooked s t a t e , mutagen content d i d not increase a p p r e c i a b l y u n t i l heated twice the recommended times (Table I I ) .

Table I I .

E f f e c t of A d d i t i o n a l Heating on Mutagenicity of Processed Seafoods 3

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch007

Product Breaded

fish

Heating Method

fillets Bake (190°C) π tt tt

Breaded

It

Deep f r y (200°C) It

Basic e x t r a c t s with S_. typhimurium

MAR (80 g Eq)

0 20 30 40 0 2 4b

1.6 2.7 2.8 5.9 0.9 0.9 2.8

b

shrimp tt

Time (min)

tt

s t r a i n 1538 p l u s 80 μΐ S9.

These times are twice the recommended heating times. Only the most e x t e n s i v e l y overheated samples (which are n e a r l y charred) began to show s i g n i f i c a n t mutagenicity. The f i n d i n g that i n general these browned foods were nonmutagenic (even though browning model systems do produce mutagens) may i n d i c a t e that the mutagens are bound to or a s s o c i a t e d with h i g h molecular compounds and-thus not e x t r a c t e d by the procedure used here. Canned foods are another c l a s s of commercially processed foods which are widely consumed by the p u b l i c . These products are subjected to extensive heat p r o c e s s i n g to achieve commercial s t e r i l i t y ; thus mutagen formation seemed to be a d i s t i n c t p o s s i b i l i t y . A v a r i e t y of canned meat, p o u l t r y , seafoods and vegetable products were t h e r e f o r e surveyed f o r mutagenicity (Tables I I I and I V ) . E x t r a c t s of raw products (beef, chicken, turkey, salmon and clams) e x h i b i t e d no mutagenicity; n e i t h e r d i d the e x t r a c t s of the vegetable products tested (tomatoes, peas, green beans, beets and corn). Beef and b e e f - c o n t a i n i n g products g e n e r a l l y possessed mutagenic a c t i v i t y while seafoods were more d i v e r s e i n t h e i r mutagenic response; canned pink salmon d i s p l a y e d the highest l e v e l s of any of the canned foods t e s t e d , tuna packed i n water and sardines were without mutagenicity. The chemical i d e n t i t y of the mutagens i n these canned products i s unknown at t h i s time. They do, however, e x h i b i t the same Salmonella s t r a i n s p e c i f i c i t y and e x t r a c t i o n behavior as mutagens found i n g r i l l e d f i s h and

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Table I I I .

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch007

Beef b r o t h Beef stew ( r e t o r t pouch) Roast beef hash C h i l i w/beef (brand #1) C h i l i w/beef (brand //2) Roast beef S.

MAR (80 g E q )

Product a

a

3.0 2.6 2.2 1.3 1.0 0.8

mix.

Mutagenicity of B a s i c E x t r a c t s from Canned Seafoods

Product

MAR (80 g E q )

Pink salmon (brand //l) Pink salmon (brand #2) Red salmon (brand #1) Red salmon (brand //2) Mackerel

17.6 11.9 8.5 7.3 7.2

a

MAR (80 g E q )

Corned beef hash Spaghetti w/meatballs Chunk turkey Chunk chicken Ham Vienna sausage

13.0 7.4 6.0 4.9 4.4 4.6

typhimurium s t r a i n TA98 with S9

Table IV.

FEEDS

Mutagenicity of B a s i c E x t r a c t s from Selected Canned Meat and P o u l t r y Products

Products

a

IN F O O D S A N D

a

S_. typhimurium s t r a i n TA98 with S9

Product Tuna ( o i l pack) Minced clams Tuna (water pack) Sardines

MAR (80 g Eq)' 3.8 3.8 1.6 1.3

mix.

beef, s e v e r a l of which have r e c e n t l y been shown to produce hepatomas when fed to r a t s and mice (19). I f the mutagens i n the canned products are a l s o i s o l a t e d , i d e n t i f i e d and found to be c a r c i n o g e n i c , they may pose a r i s k to humans because of the s i g n i f i c a n t q u a n t i t i e s of canned foods consumed. In the U.S. i n 1980 about 3 b i l l i o n pounds of meat products and 1.4 b i l l i o n pounds of seafoods were processed by canning (20). Some of the i m p l i c a t i o n s f o r the food i n d u s t r y and r e g u l a t o r y agencies i f the substances formed during canning do prove harmful w i l l be pointed out below. The N a t i o n a l Research C o u n c i l i n i t s recent report on D i e t , N u t r i t i o n and Cancer (21) urged that e f f o r t s be made to reduce or e l i m i n a t e mutagens i n foods. An understanding of the process of mutagen formation i s e s s e n t i a l to the development of methods to modify mutagen formation during canning. Two products, ground beef and pink salmon, were subjected to s e v e r a l precanning t r e a t ments to i n v e s t i g a t e the e f f e c t s on mutagen formation. Ground beef was mixed with an equal weight of d i s t i l l e d water and b o i l e d f o r 10 min. The s o l i d s were then separated from the f l u i d s (stock) by f i l t r a t i o n and p o r t i o n s of each were

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extracted immediately or placed i n cans, sealed and processed f o r 85 min at 117°C. Table V shows the r e s u l t s of mutagenicity t e s t s on the b a s i c e x t r a c t s from these samples.

Table V.

Mutagenicity of Ground Beef and Beef Stock

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch007

Precanning Treatment None Boil Boil Boil Boil

MAR

Sample Ground beef (canned) S o l i d s (not canned) S o l i d s (canned) Stock (not canned) Stock (canned)

a

_S. typhimurium s t r a i n 1538 with S9

(80 g Eq)' 3.7 0.9 1.4 3.2 9.3

mix.

Mutagens were formed when ground beef was canned. The mutagenicity decreased when the ground beef was washed with water p r i o r to canning. The wash water (stock) apparently e x t r a c t e d precursors which could form mutagens upon heating; the canned stock produced an MAR more than twice that of the canned untreated meat. Pink salmon was treated p r i o r to canning by steaming, f r e e z i n g or combined f r e e z i n g then steaming. The stock which l e f t the f l e s h during steaming (13% of the t o t a l weight) was c o l l e c t e d and a l s o canned. Table VI shows the e f f e c t s of these precanning treatments on the mutagenicity of canned pink salmon.

Table VI.

V a r i a t i o n s i n Mutagenicity of Salmon A f t e r D i f f e r e n t Precanning Treatments

Pretreatment/samples None/flesh (canned) Steam/flesh (not canned) Steam/flesh (canned) Steam/stock (not canned) Steam/stock (canned) F r e e z e / f l e s h (canned) Freeze, steam/flesh (canned) Freeze, steam/flesh (not canned) Freeze, steam/stock (canned) Freeze, steam/stock (not canned)

Revertants produced mean ± SD (n=4) 98 10 232 11 1022 270 237 18 1182 36

S^. typhimurium s t r a i n TA98 with S9 mix 23-30).

+ 24 + 7 + 37 + 14 + 89 + 32 + 25 + 32 + 240 + 48

MAR (80 g E q ) 4.3 1.3 8.7 1.4 35.0 10.0 8.9 1.6 40.4 1.2

(mean spontaneous =

a

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The steaming and/or f r e e z i n g pretreatments a l l appeared to increase the mutagenicity of the canned product. These p r e t r e a t ments would lead to d i s r u p t i o n of c e l l s w i t h i n the t i s s u e , p o s s i b l y allowing more intimate contact between the reactants during the d u r a t i o n of heat p r o c e s s i n g . During heat processing of the untreated f r e s h t i s s u e , the c e l l u l a r s t r u c t u r e a l s o i s disrupted as the heat penetrates the f l e s h , but t h i s process occurs r a t h e r slowly due to the r e s i s t a n c e to heat t r a n s f e r that e x i s t s i n a s o l i d product of t h i s type. As was the case w i t h the ground beef stock, the canned f i s h stock e x h i b i t e d higher mutagenicity than the f l e s h . However, the steaming was not n e a r l y as e f f e c t i v e i n removing the mutagen precursors from the f i s h as washing of the ground beef had been. T h i s was undoubtedly due i n p a r t to the p h y s i c a l form of the ground beef i n which much c e l l u l a r d i s r u p t i o n had taken p l a c e due merely to g r i n d i n g . The l a r g e p r o p o r t i o n of water used f o r r i n s i n g the beef was probably a l s o an important f a c t o r i n the more e f f i c i e n t removal of mutagen p r e c u r s o r s . T h i s a l s o , however, r e s u l t e d i n d i l u t i o n of the p r e c u r s o r s , e x p l a i n i n g the apparently lower mutagenicity of the beef stock compared to the f i s h stock (which was equal to only about 13% of the o r i g i n a l weight of fish). The r e s u l t s of t h i s study with canned foods b r i n g up s e v e r a l issues which have not been d e a l t with i n the past. Up to the present time, s t u d i e s d e a l i n g with mutagens i n heated foods have been concerned with mutagens formed during the cooking process. In that case, the consumer has some c o n t r o l over how he cooks h i s food and has a choice of methods which can reduce or e l i m i n a t e mutagen production. For example, he can use low temperature techniques such as microwaving or steaming r a t h e r than f r y i n g of grilling. However, the consumer has no choice i n the case of canned foods where the only way to avoid these substances i s to not purchase the product. I f the compounds i n canned foods are indeed c a r c i n o g e n i c , some questions that need to be addressed are, "Should these compounds be regulated or should the foods be regulated because of a p o s s i b l e h e a l t h hazard? I f they are to be regulated, what p r o v i s i o n s of the current r e g u l a t i o n s apply?" The f i r s t l e g i s l a t i o n that comes to mind i s the Delaney clause because i t deals with carcinogens, but when the clause i s c l o s e l y examined one f i n d s that i t deals only with food a d d i t i v e s . Thermal processing adds nothing to the product other than heat. Heat processing could p o s s i b l y be compared to food i r r a d i a t i o n i n that both are d i f f e r e n t types of food processing techniques and food i r r a d i a t i o n i s considered an " a d d i t i v e " by FDA and subject to i t s r e g u l a t i o n s on a d d i t i v e s . However, heat or thermal processing i s not considered an a d d i t i v e , t h e r e f o r e , the mutagens could a l s o not be d e a l t with under Section 409 of the Food, Drug and Cosmetic Act which deals with unsafe food a d d i t i v e s , or S e c t i o n 406 of the same Act which

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Formation

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Foods

deals with tolerances f o r added poisonous i n g r e d i e n t s i n foods. These mutagens (or carcinogens) a l s o probably can't be considered a carcinogenic " c o n s t i t u e n t " as defined i n the FDA s l a t e s t " c o n s t i t u e n t s " p o l i c y where a carcinogenic impurity i s allowed to be present with a non-carcinogenic a d d i t i v e . The s e c t i o n where mutagens/carcinogens formed as a r e s u l t of processing p o s s i b l y could be regulated i s i n Section 402 of the Food, Drug and Cosmetic Act d e a l i n g with a d u l t e r a t e d food. This s e c t i o n says t h a t : "...A food s h a l l be deemed to be a d u l t e r a t e d i f i t bears or contains any poisonous or d e l e t e r i o u s substances which may render i t i n j u r i o u s to h e a l t h ...". One o r g a n i z a t i o n , the C o u n c i l f o r A g r i c u l t u r a l Science and Technology (CAST), i n i t s r e p o r t No. 89 (22), decided to c a t e g o r i z e n a t u r a l l y o c c u r r i n g carcinogens and carcinogens formed during cooking under t h i s Section 402. Apparently, however, i f these carcinogens ( n a t u r a l l y o c c u r r i n g or formed) are present i n minute concentrations, CAST does not consider them to c o n s t i t u t e a t h r e a t to p u b l i c h e a l t h which would j u s t i f y a c t i o n against them. The FDA has stated that i n r e g u l a t i n g n a t u r a l p o t e n t i a l hazardous c o n s t i t u e n t s , i t would consider the b e n e f i t s of the food and the impact a p r o h i b i t i o n would have on the a v a i l a b l e food supply. The FDA a l s o stated that they would not remove from the food supply a d e l e t e r i o u s substance that i s a n u t r i e n t , widely used and entrenched i n the food system unless i t posed a s i g n i f i c a n t h e a l t h r i s k (23). The meaning of the phrases, " i n j u r i o u s to h e a l t h " i n S e c t i o n 402 of the Food, Drug and Cosmetic Act and " s i g n i f i c a n t h e a l t h r i s k " mentioned above are vague i n reference to occurrences or formation of mutagens i n foods and must be c l a r i f i e d before meaningful d e c i s i o n s can be made. For example, does a s i g n i f i cant h e a l t h r i s k " e x i s t i f these mutagens are determined to be mammalian carcinogens but found i n low l e v e l s i n canned foods? I t c l e a r l y appears that our current r e g u l a t i o n s have d i f f i c u l t y i n c o n s i s t e n t l y d e a l i n g with low and u n c e r t a i n r i s k s such as we f i n d i n canned foods and that we do need a change i n the way we evaluate p o t e n t i a l hazards i n foods. The Food Safety Council's proposed food s a f e t y e v a l u a t i o n process (24), i n which a systematic sequence i s followed f o r e v a l u a t i o n of the s a f e t y of a food component, may be a major step i n that d i r e c t i o n . T

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125

Acknowledgments T h i s research was supported i n p a r t by the U n i v e r s i t y of Washington Sea Grant O f f i c e , I n s t i t u t e f o r Food Science and Technology and the Egtvedt Food Research Fund.

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Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch007

Literature Cited 1. Lijinsky, W.; Shubik, P. Science 1964, 145, 53-55. 2. Howard, J.W.; Tesque, R.T.; White, R.H.; Fry, B.E. J . Am. Oil Chem. Soc. 1966, 49, 596-601. 3. Nagao, M.; Honda, M.; Seino, Y.; Yahagi, T.; Sugimura, T. Cancer Lett. 1977, 2, 221-26. 4. Sugimura, T. "Naturally Occurring Carcinogens-Mutagens and Modulators of Carcinogenesis"; Miller, E.C.; Miller, J.Α.; Hirono, I.; Sugimura, T.; Takayama, S., Eds.; University Park Press: Baltimore, 1979, p. 241. 5. Yamamoto, T.; Tsuji, K.; Kosu, T.; Okamoto, T.; Shudo, K.; Takeda, K.; Iitaka, Y.; Yamaguchi, K.; Seino, Y.; Yahagi, T.; Nagao, M.; Sugimura, T. Proc. Jpn. Acad. 1978, 54B, 248-50. 6. Kasai, H.; Yamaizumi, Z.; Wakabayashi, Κ.; Nagao, M.; Sugimura, T.; Yokoyama, S.; Miyazawa, T.; Spingarn, N.E.; Weisburger, J.H.; Nishimura, S. Proc. Jpn. Acad. 1980, 54B, 278-83. 7. Kasai, H.; Yamaizumi, Z.; Shiomi, T.; Yokoyama, S.; Miyazawa, T.; Wakabayashi, Κ.; Nagao, M.; Sugimura, T.; Nishimura, S. Chem. Lett. 1981, 485-88. 8. Bjeldanes, L.F.; Morris, M.M.; Felton, J.S.; Healy, S.; Stuermer, D.; Berry, P.; Timourian, H.; Hatch, F.T. Fd. Chem. Toxicol. 1982, 20, 357-69. 9. Pariza, M.W.; Ashoor, S.H.; Chu, F.S.; Lund, D.B. Cancer Lett. 1979, 7, 63-69. 10. Krone, C.A.; Iwaoka, W.T. Cancer Lett. 1981, 14, 93-99. 11. Spingarn, N.E.; Garvie, C.T. J. Agric. Food Chem. 1979, 27(1), 1319-21. 12. Mihara, S.; Shibamoto, T. J. Agric. Food Chem. 1980, 28(1), 62-66. 13. Powrie, W.D.; Wa, C.H.; Rosin, M.P., Stich, H.F. J. Food Sci. 1981, 46, 1433-38. 14. Stich, H.F.; Stich, W.; Rosin, M.P.; Powrie, W.D. Mutation Res. 91, 129-136. 15. Spingarn, N.E.; Slocum, L.A.; Weisburger, J.H. Cancer Lett. 1980, 9, 7-12. 16. Iwaoka, W.T.; Krone, C.A.; Sullivan, J . J . ; Meaker, E.H.; Johnson, C.A.; Miyasato, L.S. Cancer Lett. 1981, 11, 225-230. 17. Pariza, M.W.; Ashoor, S.H.; Chu, F.S. Food Cosmet. Toxicol. 1979, 17, 429-30. 18. Ames, B.N.; McCann, J.; Yamasaki, E. Mutation Res. 1975, 31, 347-364. 19. Sugimura, T. Cancer 1982, 49(10), 1970-84. 20. "The Almanac of the Canning, Freezing and Preserving Industries," Edward E. Judge and Sons: Westminster, Maryland, 1981. 21. "Diet, Nutrition and Cancer", National Research Council, National Academy Press: Washington, D.C. 1982.

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Council for Agricultural Science and Technology. Report No. 89. "Regulations of Potential Carcinogens in the Food Supply"; Ames, Iowa, June 1981. 23. "Regulations of Cancer Causing Food Additives - Time for a Change", Report to Congress of the U.S., Comptroller General, December 1981. 24. Food Safety Council. "A Proposed Food Safety Evaluation Process"; Nutrition Foundation, Inc.: Washington, D.C. 1982, p. 7-8.

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch007

RECEIVED June 28,

1983

8 Biological Properties of Heated Dietary Fats

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch008

J. C. ALEXANDER Department of Nutrition, College of Biological Science, University of Guelph, Guelph, Ontario, Canada N1G 2W1 Thermal oxidation of dietary fats, with the formation of potentially toxic derivatives during heating and processing, is related to the conditions used in the home and the food service industry. During deep-fat frying many volatile and non-volatile compounds are formed, some of which can be toxic depending on the level of intake. Many chemical and biological studies have been carried out, and experimental findings indicate that possible dietary hazard should be greater as the severity of the treatment of the fat is increased. Observations with animals fed these fats have shown adverse effects ranging from depression in growth, diminished feed efficiency, increased liver size, fatty necrosis of the liver and numerous other organ lesions. Specific effects on biological tissues can be verified by selected techniques including histopathological evaluations, biochemical parameters, and tissue culture in monolayers. Fractionation of heated fat samples serves to concentrate a number of the unnatural components, and incorporation of these materials into rat diets has enabled experimenters to observe distinct reactions by the animals. Practical processing and frying operations usually produce low levels of nutritionally undesirable products, but it is worthwhile to recognize their possible adverse biological effects. The b i o l o g i c a l p r o p e r t i e s of thermally o x i d i z e d f a t s have been studied f o r many years. E v a l u a t i o n of laboratory-heated and commercially-used f a t s i n d i e t s f o r animals have included feed consumption, weight g a i n , and feed e f f i c i e n c y (1-4), pathology (1-2)9 organ weights (4, 10), enzyme assays (11), and t o t a l l i p i d 0097-6156/83/0234-0129$06.00/0 © 1983 A m e r i c a n C h e m i c a l S o c i e t y

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch008

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content and t i s s u e f a t t y a c i d composition (12-15). A v a r i e t y of thermally o x i d i z e d f a t s was included and r e s u l t s ranged from m i l d responses to s u b s t a n t i a l adverse e f f e c t s on the animals. Ques­ t i o n s remain regarding the p o t e n t i a l t o x i c i t y of l a b o r a t o r y heated or commercially used f r y i n g f a t s . Evidence i n d i c a t e s that secondary o x i d a t i o n products such as monomeric, dimeric and polymeric compounds accumulate i n the heated f a t s and may subsequently be ingested with the f r i e d foods. Johnson et a l . (16) observed that thermally o x i d i z e d o i l i s not so r a p i d l y hydrolyzed as the corresponding unheated o i l . Nolen (5) found when feeding male dogs d i e t s c o n t a i n i n g a 15% l e v e l of f r e s h or heated p a r t i a l l y hydrogenated soybean o i l that the heated f a t reduced the a b s o r p t i o n , growth r a t e and feed e f f i c i e n c y . These products r a t h e r than peroxides are the p r i n c i p a l f a c t o r s i n ad­ verse e f f e c t s seen with thermally o x i d i z e d f a t s . Paik et a l . (17) studied mice dosed with methyl l i n o l e a t e hydroperoxides or a u t o x i dized methyl l i n o l e a t e c o n t a i n i n g secondary o x i d a t i o n products. M o r t a l i t y was 50% and 100% r e s p e c t i v e l y . Congestion i n t i s s u e s , f a t t y degeneration and n e c r o s i s were observed, and the amount of impairment c o r r e l a t e d with the type of m a t e r i a l f e d . The c o n c l u ­ sions were confirmed by h i s t o p a t h o l o g i c a l o b s e r v a t i o n s , and v o l a ­ t i l e low molecular weight compounds c o n t a i n i n g carbonyl groups were suspected of being i n v o l v e d . The r e a c t i v i t y of f a t t y a c i d s increases with the degree of u n s a t u r a t i o n , but the d i s t r i b u t i o n and geometry of double bonds a l s o i n f l u e n c e the extent of o x i d a ­ t i o n (18). Double bonds become conjugated or l o s t as they are involved i n r e a c t i o n s forming v a r i o u s secondary products, some of which have been i d e n t i f i e d as c y c l i c compounds, s c i s s i o n products, dimers and l a r g e r molecules (19-23). The s e v e r i t y of c o n d i t i o n s ( a e r a t i o n , temperature and heating time) p l a y s an important r o l e i n the degree of d e t e r i o r a t i o n of f r y i n g f a t s ( 1 ) . Perkins and Kummerow (24) confirmed the e a r l i e r observations of Crampton et a l . (25) that the non-urea-adductable p o r t i o n of heated f a t s was most t o x i c to animals. These concentrates, c o n t a i n i n g monomeric and dimeric d e r i v a t i v e s , are more t o x i c than the l a r g e r polymers due to b e t t e r a b s o r p t i o n 02, 10, 26). Urea f i l t r a t e of heated f a t , fed to r a t s , r e s u l t e d i n a 30% r e d u c t i o n i n the o x i d a t i o n of p a l ­ m i t i c a c i d to CO2 (27). Animals given low l e v e l s of vitamins and thermally o x i d i z e d f a t s i n t h e i r d i e t a t a f i x e d p r o t e i n concen­ t r a t i o n responded p o o r l y compared to those which received f r e s h f a t s and the same amount of vitamins ( 3 ) . A r e a l p o s i t i v e r e ­ sponse was obtained i n the presence of the heated f a t when the p r o t e i n l e v e l was i n c r e a s e d . Tappel (28) suggested that chemical d e t e r i o r a t i v e e f f e c t s due to f r e e r a d i c a l s produced i n heated f a t s (hydroxyl and hydroperoxyl) might be slowed by increased amounts of d i e t a r y a n t i o x i d a n t s . Fukuzawa and Sato (29) s e l e c t e d 12-ketoo l e i c a c i d as a degradation product of l i p i d p e r o x i d a t i o n and compared i t s e f f e c t s on r a t t i s s u e s with those of a v i t a m i n Ε d e f i c i e n c y . Both increased f l u o r e s c e n t production i n the l i v e r , as w e l l as hemolysis and plasma a l k a l i n e phosphatase l e v e l i n the blood.

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131

Because of the s e v e r i t y of some problems reported when experimental animals are fed compounds from thermally o x i d i z e d food f a t s there i s j u s t i f i e d concern about e f f e c t s some of these d e r i v a t i v e s could have on consumers. E a r l y work which showed adverse e f f e c t s on animals was complicated by improper p r o t e c t i o n o f the d i e t leading to v i t a m i n d e f i c i e n c i e s . However, i n studies with used f r y i n g f a t s , Alexander (30) found that frequent d i e t prepar a t i o n and f e e d i n g , and the use of a n t i o x i d a n t s and r e f r i g e r a t i o n could avoid these d i f f i c u l t i e s . In recent years, a number of w e l l conducted studies have produced considerable evidence that o x i d i z e d and abused f r y i n g f a t s c o n t a i n p o t e n t i a l l y t o x i c constituents.

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch008

Experimental To evaluate the b i o l o g i c a l e f f e c t s of thermally o x i d i z e d f a t s , i s o l a t e d concentrates of f a t t y a c i d d e r i v a t i v e s were prepared. Fats used were corn o i l (CO) o l i v e o i l (00) low e r u c i c a c i d rapeseed o i l (LE) and l a r d (LA). The heating c o n d i t i o n s were those of G a b r i e l e t a l . Ç7). Each f a t was heated i n a s t a i n l e s s s t e e l beaker f o r 72 h r a t a c o n t r o l l e d temperature of 180°C. Each day i t was s t i r r e d continuously w i t h a mechanical s t i r r e r f o r 12 h r and by hand every h r f o r 12 h r to ensure aera t i o n and mixing. D i s t i l l a t i o n and urea treatment concentrated the c y c l i c and branched chain degradation products. Intubation experiments with r a t s were used as short-term studies to d e t e r mine the t o x i c i t y of the d i s t i l l a b l e non-urea-adductable (DNUA) f r a c t i o n s obtained from e t h y l esters produced from thermally o x i dized f a t s (31,32). A urea adduction method modified from that of Eisenhauer and Beal (33) was used to produce the DNUA concent r a t e s . A l l r a t s were intubated d a i l y with 0.5 ml of e i t h e r DNUA m a t e r i a l s from thermally o x i d i z e d f a t s or the r e s p e c t i v e f r e s h f a t s , using a rubber stomach tube (no. 8 catheter) on a s y r i n g e . Body weights were recorded d a i l y , and the animals were examined on a r e g u l a r b a s i s to detect e a r l y symptoms o f t o x i c i t y . After approximately three days, due to m o r b i d i t y , the animals were k i l l e d . H i s t o p a t h o l o g i c a l examination o f the h e a r t , l i v e r and kidneys o f the r a t s was c a r r i e d out to q u a n t i t a t e t i s s u e damage, and l e s i o n s i n the organs were graded as described by G a b r i e l et a l . ( 7 ) . Organ weights were recorded and p o r t i o n s were frozen i n l i q u i d n i t r o g e n f o r l i p i d a n a l y s i s (34) . F a t t y a c i d methyl e s t e r s were prepared from the n e u t r a l and p o l a r l i p i d f r a c t i o n s of the organs (35,36) and analyzed f o r component f a t t y acids by GLC. Types of l e s i o n s found i n the t i s s u e s e c t i o n s were graded as to incidence and s e v e r i t y on a s c a l e from zero f o r normal, to three f o r n e c r o t i c t i s s u e , i n the f o l l o w i n g anatomical s t r u c t u r e s : Heart: Myocardial n u c l e i N u c l e i of v a s c u l a r media and endothelium I n t e r s t i t i a l t i s s u e and myofibers

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Liver :

XENOBIOTICS

IN FOODS A N D F E E D S

Hepatocellular nuclei H e p a t o c e l l u l a r cytoplasm Kupffer and e n d o t h e l i a l c e l l s Kidney : Glomeruli Tubules V e s s e l s and i n t e r s t i t i a l t i s s u e s Animal t i s s u e c e l l s grown i n c u l t u r e medium i n the form of a monolayer are a u s e f u l b i o l o g i c a l l i v i n g model to observe p h y s i o ­ l o g i c a l , morphological or metabolic changes i n the presence of conpounds added to the medium. B i r d and Alexander (15, 37) r e ­ ported on e f f e c t s of thermally o x i d i z e d corn o i l and o l i v e o i l on i n v i t r o heart c e l l s . The DNUA from f a t thermally o x i d i z e d as described e a r l i e r (_7) was i s o l a t e d . Free f a t t y a c i d s from the f r e s h f a t c o n t r o l s o r from the DNUA of the heated f a t s were pre­ pared (38). Primary c u l t u r e s of neonatal h e a r t c e l l s were prepared from 2-5 day o l d r a t s by the method of Rogers (39) as monolayered c o v e r s l i p c u l t u r e s . The h e a r t s were e x c i s e d a s e p t i c a l l y and t r a n s f e r r e d to a p e t r i d i s h c o n t a i n i n g phosphate-buffered s a l i n e . The t i s s u e was chopped f i n e l y and separated i n t o s i n g l e c e l l s by t r y p s i n i z a t i o n using 0.25% t r y p s i n s o l u t i o n (15). Leighton tubes c o n t a i n i n g a c u l t u r e medium s u p p l i e d w i t h 5% f e t a l c a l f serum were seeded with 2 χ 10^ c e l l s / m l o f medium. Four day o l d c u l t u r e s were exposed to f r e s h and heated f a t t y a c i d f r a c t i o n s (60 or 100 yg/ml) i n the form of an emulsion with bovine serum albumin d i s ­ solved i n phosphate-buffered s a l i n e (PBS). The r e q u i r e d concen­ t r a t i o n of each f r e e f a t t y a c i d f r a c t i o n , d i s s o l v e d i n hexane, was t r a n s f e r r e d to a 100 ml s t e r i l i z e d b o t t l e , and the s o l v e n t was evaporated completely. The s o l u t i o n of bovine serum albumin (40 mg/ml of PBS) was added to the l i p i d f r a c t i o n s . A r a t i o of f r e e f a t t y a c i d f r a c t i o n to bovine serum albumin of 1:60 (w/w) was maintained. The b o t t l e was screw-capped and incubated a t 40° with o c c a s i o n a l shaking f o r two h r . T h i s i n c u b a t i o n was s u f f i ­ c i e n t to o b t a i n an emulsion o f f r e e f a t t y a c i d s ready f o r admin­ i s t r a t i o n i n t o the c u l t u r e medium (15). C e l l u l a r l i p i d was e x t r a c t e d by means of the procedure of Folch e t a l . (40) and f r a c t i o n a t e d by t h i n l a y e r chromatography (TLC). Glass p l a t e s coated with S i l i c a Gel G of 0.5 ml thickness and a solvent system c o n t a i n i n g heptane/isopropyl e t h e r / a c e t i c a c i d (60:40:3) were used (41). C u l t u r e s of heart c e l l s were ob­ served p e r i o d i c a l l y with a Nikon i n v e r t e d microscrope with phase c o n t r a s t o p t i c s . For d e t a i l e d morphology, r e p l i c a t e c u l t u r e s a l s o were s t a i n e d with May-Grunwald-Giemsa s t a i n (42). I n t r a ­ c e l l u l a r l i p i d accumulation was assessed s u b j e c t i v e l y with the phase c o n t r a s t microscope, and a numerical s c a l e ranging from zero to four was used. The zero value was given to c e l l s showing no l i p i d d r o p l e t s , as i n those grown i n the medium c o n t a i n i n g 5% f e t a l bovine serum without a d d i t i o n a l l i p i d . A v a l u e of four was given to a f i e l d c o n s i s t i n g of c e l l s e x h i b i t i n g abundant f a t d r o p l e t s where most of the e x t r a n u c l e a r space appeared to be occupied with l i p i d (38).

8.

ALEXANDER

Biological

Properties

of Heated

Dietary

133

Fats

Stained cover s l i p c u l t u r e s from each treatment group were observed under the x40 o b j e c t i v e t o estimate the e f f e c t o f v a r i o u s treatments on growth p a t t e r n and c e l l morphology. Twenty random f i e l d s were s e l e c t e d , and the number o f c e l l s i n each f i e l d , the number of c e l l s i n v a r i o u s stages of m i t o s i s , and the number of pyknotic n u c l e i were recorded. The m i t o t i c index and percent pyknotic c e l l s were c a l c u l a t e d as percentages based on the t o t a l number of c e l l s observed i n 20 random f i e l d s and were not average values of r e p l i c a t i o n s . The c e l l u l a r p r o t e i n was q u a n t i t a t e d by the method o f Lowry e t a l . (43) as modified by Oyama and Eagle (44).

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch008

Results Intubation of the r a t s with three of the heated f a t DNUA samples, heated o l i v e o i l (HOO), heated l a r d (HLA) and heated low e r u c i c a c i d rapeseed o i l (HLE) r e s u l t e d i n e a r l y signs of physiol o g i c a l s t r e s s . Within 10 h r the animals became d i s o r i e n t e d and nervous and feed and water consumption were reduced. A l o s s of body weight was observed. Rats given DNUA from heated corn o i l (HCO) were i n d i s t i n g u i s h a b l e from those given the f r e s h f a t s (Table I ) . Average weight o f the animals a t the s t a r t o f the experiment was 59 g.

Table I . Body Weights of Rats Intubated with D i f f e r e n t D i e t a r y Fats D i e t a r y Fat

Body Weight (g)

1

a

CO HCO

75 70

00 H00

b 49

LE HLE

62 48

LA HLA

71 49

a

7 0

b

a

b

a

b

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LU

Û

10

05

1

C O R N OIL Figure DNUA

OLIVE OIL

1. Histological scores for organs of rats intubated with fresh fats (O) or of heated fats (%). (Reproduced with permission from Ref 31. Copyright 1979, Geron-X Inc.)

135

XENOBIOTICS

136

IN F O O D S A N D F E E D S

2 5ρ 20

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch008

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-

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0

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LU OC

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Ο υ

20

LEAR

LARD

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LARD

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Figure DNUA

2. Histological scores for organs of rats intubated with fresh fats (O) or of heated fats (·). (Reproduced with permission from Ref 32. Copyright 1979, Geron-X Inc.)

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch008

8.

ALEXANDER

Biological

Properties

of Heated

Dietary

Fats

137

Figure 3. Heart of rat intubated with DNUA-HLA. Blood vessel wall has lost its integrity, and endomysium is very prominent. Hematoxylin and eosin, x 750. (Reproduced with permission from Ref. 32. Copyright 1979, Geron-X Inc.)

Figure 4. Liver of rat intubated with DNUA-HLA. Pyknosis and fragmentation of necrotic hepatocytes. Hematoxylin and eosin, x500. (Reproduced with permission from Ref. 32. Copyright 1979, Geron-X Inc.)

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch008

138

XENOBIOTICS

IN F O O D S A N D

Figure 5. Kidney of rat intubated with DNUA-HLE. Cellular and granular fill lumina of uriniferoustubules. Hematoxylin and eosin, x344. (Reproduced permission from Ref. 32. Copyright 1979, Geron-X Inc.)

FEEDS

casts with

8.

ALEXANDER

Biological

Table I I I .

Properties

of Heated

Dietary

Fats

139

R e l a t i v e Organ Weights and T o t a l Organ L i p i d s (LEAR and LARD) 1

Dietary

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch008

Fat

Heart

Liver

Lipid (%)

Wt. (%)

LE HLE

0.47 0.44

LA HLA

0.41 0.42

3

a

a

a

b

5.2 9.3

a

b

4.7 7.7

a

Kidney

Lipid (%)

Wt. (%) b

4.4 7.0

a

b

4.4 8.8

a

7.9 7.8

a

a

b

5.9 9.5

a

Lipid (%)

Wt. (%) 1.2 1.4

ab

a

7.4 5.9

a

a

6.9 6.7

b

1.0 1.6

a

a

e

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Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch009

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(D Ό •H 14, 15). S e v e r a l p r o c e d u r e s a r e a v a i l a b l e ranging from t h e highly s o p h i s t i c a t e d mass spectrometric (14) t o a s i m p l e r a p i d "go o r no g o " t e s t t h a t can be u s e d a t t h e pTant o r e l e v a t o r site (15). Many U.S. a n d C a n a d i a n c o m p a n i e s a r e a l r e a d y t e s t i n g f o r v o m i t o x i n , some a s p a r t o f t h e i r own q u a l i t y c o n t r o l p r o g r a m , o t h e r s b e c a u s e o f t h e C a n a d i a n g u i d e l i n e s which a l l o w a maximum l e v e l o f 0.1 ppm v o m i t o x i n i n wheat f l o u r o r b r a n u s e d t o make s t a p l e f o o d p r o d u c t s ( e . g . , b r e a d ) a n d 0.3 ppm i n t h e f l o u r o r b r a n u s e d t o make n o n - s t a p l e f o o d p r o d u c t s ( e . g . , cakes, cookies, p r e t z e l s , e t c . ) . A t the present time, there are no U.S. g u i d e l i n e s a s s u c h , b u t t h e Food and Drug A d m i n i s t r a t i o n has d e v e l o p e d " a d v i s o r y " l e v e l s f o r v o m i t o x i n i n wheat a n d wheat p r o d u c t s . The a d v i s o r y l e v e l f o r wheat a n d wheat p r o d u c t s i n t e n d e d f o r human c o n s u m p t i o n i s 2 ppm i n t h e wheat b e f o r e m i l l i n g and 1 ppm i n t h e f i n i s h e d wheat p r o d u c t s . F o r wheat a n d wheat p r o d u c t s which a r e u s e d a s a n i m a l f e e d i n g r e d i e n t s , t h e a d v i s o r y l e v e l i s 4 ppm w i t h t h e a d d e d r e c o m m e n d a t i o n t h a t t h e s e i n g r e d i e n t s n o t e x c e e d 1 0 % o f swine a n d p e t d i e t s o r 50% o f t h e d i e t s o f r u m i n a n t s a n d p o u l t r y . T h e r e a s o n f o r t h e l o w e r recommended l e v e l f o r s w i n e , c a t s , a n d dogs compared t o r u m i n a n t s a n d p o u l t r y i s t h a t t h e f o r m e r a r e more s u s c e p t i b l e t o t h e known e f f e c t s o f v o m i t o x i n ( r e f u s a l a n d v o m i t i n g ) . S i n c e v o m i t o x i n c a n a l s o be found i n corn a t c o n c e n t r a t i o n s a s high o r higher than i s found in wheat a n d s i n c e c o r n i s n o r m a l l y u s e d a t h i g h e r t h a n a 1 0 % l e v e l i n swine f e e d a n d p e t f o o d s , i t i s a d v i s a b l e t o c o n s i d e r a

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch015

15.

ROMER

The

Vomitoxin

Story

245

f i n a l l e v e l o f 1 ppm v o m i t o x i n i n h i g h - c o r n swine and p e t d i e t s a s a maximum c o n c e n t r a t i o n and m o n i t o r t h e c o r n a c c o r d i n g l y . S e v e r a l f e e d i n g s t u d i e s have been p e r f o r m e d o n swine u s i n g , i n most c a s e s , c o r n o r wheat n a t u r a l l y c o n t a m i n a t e d w i t h v o m i t o x i n ( 4 , 1 6 , 2 7 ) . I n g e n e r a l , t h e o n l y e f f e c t n o t i c e d was l o w e r e d f e e d c o n s u m p t i o n w i t h swine when 0.3 ppm o r more v o m i t o x i n was i n c o r p o r a t e d i n t o t h e d i e t v i a n a t u r a l l y c o n t a m i n a t e d wheat o r c o r n . T h e l o w e r e d f e e d c o n s u m p t i o n was n o t i c e d m a i n l y d u r i n g t h e f i r s t week o f t h e s t u d i e s which r e s u l t s i n a d e p r e s s i o n i n f i n a l w e i g h t compared t o c o n t r o l s . When p u r e v o m i t o x i n was i n c o r p o r a t e d i n t o swine d i e t s , t h e f e e d c o n s u m p t i o n d e c r e a s e r a n g e d f r o m 20% a t 3.6 ppm t o 90% a t 40 ppm. The minimum e m e t i c dose was 0.1 t o 0.2 mg/kg body w e i g h t when a d m i n i s t r a t i o n was o r a l ( g a v a g e ) ( 1 8 ) . I t i s n e c e s s a r y t o remember t h a t some o f t h e lowered f e e d consumption in d i e t s c o n t a i n i n g n a t u r a l l y contami n a t e d i n g r e d i e n t s may be c a u s e d b y t r i c h o t h e c e n e m y c o t o x i n s o t h e r t h a n v o m i t o x i n w h i c h have n o t y e t been i d e n t i f i e d . P o u l t r y have been f o u n d t o be h i g h l y r e s i s t a n t t o t h e e f f e c t s of vomitoxin. A d i e t c o n t a i n i n g s u f f i c i e n t n a t u r a l l y contami n a t e d c o r n t o p r o v i d e a 50 ppm v o m i t o x i n c o n c e n t r a t i o n was f e d t o 6 day o l d b r o i l e r c o c k e r e l s f o r 6 d a y s w i t h no e f f e c t on growth o r f e e d c o n s u m p t i o n . The o n l y o b s e r v e d e f f e c t s o f t h e c o n t a m i n a t e d d i e t were p l a q u e s i n t h e mouth and g i z z a r d e r o s i o n ( 1 9 ) . When a d i e t c o n t a i n i n g 15 ppm p u r e v o m i t o x i n was f e d t o b r o i l e r s f o r 6 weeks, no d e l e t e r i o u s e f f e c t s were n o t i c e d ( 2 0 ) . A d i e t c o n t a i n i n g s u f f i c i e n t n a t u r a l l y contaminated corn t o p r o v i d e a f i n a l c o n c e n t r a t i o n o f 50 ppm v o m i t o x i n was f e d t o l a y i n g hens f o r 4 weeks w i t h no e f f e c t s o n p r o d u c t i o n , consumpt i o n , body w e i g h t , egg w e i g h t o r s h e l l s t r e n g t h . Sample c h i c k s were n e c r o p s i e d a t h a t c h and grown t o one week o f age; no t r e a t ment e f f e c t s were f o u n d ( 2 1 ) . However, i n a n o t h e r s t u d y ( 2 2 ) , when d i e t s c o n t a i n i n g 0.3 t o 0.7 ppm v o m i t o x i n f r o m n a t u r a l l y c o n t a m i n a t e d wheat were f e d t o l a y i n g hens f o r 3 weeks, t h e r e was a 2% d e c r e a s e i n egg w e i g h t and egg s h e l l t h i c k n e s s . T h i s l a t t e r r e s u l t p o i n t s o u t a s i g n i f i c a n t f a c t o r t h a t a l w a y s n e e d s t o be c o n s i d e r e d when f e e d i n g g r a i n c o n t a i n i n g v o m i t o x i n . J u s t b e c a u s e the l e v e l o f vomitoxin in the g r a i n i s not s u f f i c i e n t t o cause a p r o b l e m i n t h e a n i m a l s b e i n g f e d ( e . g . , l a y i n g h e n s ) does n o t mean t h e g r a i n i s n o t h a r m f u l . Even i f a l l o f t h e mold t o x i n s f o r w h i c h t h e r e a r e a n a l y t i c a l methods have n o t been d e t e c t e d , g r a i n c o u l d s t i l l c o n t a i n u n i d e n t i f i e d mold t o x i n s . More t h a n 30 compounds have been d i s c o v e r e d t h a t b e l o n g t o t h e t r i c h o t h e c e n e f a m i l y o f m y c o t o x i n s w h i c h i n c l u d e v o m i t o x i n , T-2 t o x i n and d i a c e t o x y s c i r p e n o l . However, t h e s e t h r e e a r e t h e o n l y t r i c h o t h e c e n e s f o r w h i c h t h e r e e x i s t r o u t i n e a n a l y t i c a l methods. The f a c t t h a t v o m i t o x i n i s f o u n d i n a sample o f g r a i n i s p r o o f t h a t a t l e a s t one mold has been g r o w i n g i n t h e g r a i n . More t h a n one mold may have been g r o w i n g and more t h a n one t o x i n may have been p r o d u c e d . C a t t l e , l i k e p o u l t r y , seem t o be u n a f f e c t e d by v o m i t o x i n . S t e e r s and h e i f e r s were f e d d i e t s c o n t a i n i n g 11 ppm v o m i t o x i n f o r

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch015

246

X E N O B I O T I C S IN F O O D S A N D

FEEDS

18 weeks w i t h no i l l e f f e c t s n o t i c e d (4J. However, n e i t h e r t h e serum c h e m i s t r y n o r t h e h i s t o p a t h o l o g y p o r t i o n o f t h i s r e s e a r c h has been c o m p l e t e d . In a n o t h e r s t u d y (23), d r y d a i r y c a t t l e were f e d a d i e t c o n t a i n i n g 4.7 ppm v o m i t o x i n f o r 30 d a y s . No e f f e c t s on f e e d c o n s u m p t i o n o r o t h e r p e r f o r m a n c e f a c t o r s were n o t i c e d . In b o t h o f t h e s e c a t t l e s t u d i e s , n a t u r a l l y c o n t a m i n a t e d wheat was the source o f vomitoxin i n the feed. We need t o r e a l i z e t h a t none o f t h e v o m i t o x i n t o x i c i t y s t u d i e s d i s c u s s e d above t e s t e d f o r i m m u n o s u p p r e s s i o n . A l s o , none o f t h e swine o r c a t t l e s t u d i e s t e s t e d f o r r e p r o d u c t i v e e f f e c t s . One o f t h e s t u d i e s c i t e d above (7) f o u n d t h a t v o m i t o x i n c a n p r o duce r e p r o d u c t i v e e f f e c t s i n l a b o r a t o r y a n i m a l s . Z e a r a l e n o n e , a m y c o t o x i n known t o p r o d u c e r e p r o d u c t i v e e f f e c t s on swine (24) a n d l e n g t h e n t h e i r e s t r u s c y c l e (25), i s o f t e n f o u n d a l o n g w i t T F v o m i t o x i n i n contaminated g r a i n . Thus, i t i s important t h a t vomitoxin be t e s t e d b o t h a l o n e a n d i n c o m b i n a t i o n w i t h z e a r a l e n o n e t o d e t e r m i n e i f i t a d v e r s e l y e f f e c t s t h e r e p r o d u c t i v e s y s t e m o f swine and/or c a t t l e . When t h e r e s u l t s o f s u b c h r o n i c a n d c h r o n i c s t u d i e s on t h e r e p r o d u c t i v e , immunosuppressive, and o t h e r p o s s i b l e adverse e f f e c t s o f v o m i t o x i n have been c o m p l e t e d , i t w i l l be e a s i e r t o j u d g e how much o f a h a z a r d v o m i t o x i n p o s e s f o r t h e f e e d a n d t h e food i n d u s t r i e s .

Literature Cited 1. Scott, P. M. Joint Mycotoxin Committee Report, Association of Official Analytical Chemists Convention, Washington, D.C., October 23, 1980. 2. Scott, P. M. Joint Mycotoxin Committee Report, Association of Official Analytical Chemists Convention, Washington, D.C., October 22, 1981. 3. Buck, W. University of Illinois, Urbana, Illinois, Personal Communication. 4. Schneider, N. R.; Carlson, M. University of Nebraska, Lincoln, Nebraska, Personal Communication. 5. Rottinghaus, G. University of Missouri, Columbia, Missouri, Personal Communication. 6. Eppley, R. M. Bureau of Foods, Food and Drug Administration, Washington, D.C., Personal Communication. 7. Khera, K. S.; Whalen, C.; Angers, G.; Vesonder, R. F.; Kuiper-Goodman, T. Bull. Environ. Contam. Toxicol., 1982, 29, 487. 8. Morrissey, R. E. To be published in April, 1983, Teratology. 9. Marasas, W. F. O.; van Rensburg, S. J.; Mirocha, C. J. J. Agric. Food Chem., 1979, 27, 5, 1108. 10. Sato, N.; Ueno, Y.; Enomoto, M. Japan. J. Pharmacol., 1975, 25, 263. 11. Scott, P. M.; Lau, P. Y.; Kanhere, S. R. JAOAC, 1981, 64, 1364 (A GLC Method).

15.

12. 13. 14. 15. 16.

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch015

17. 18. 19. 20. 21. 22. 23. 24. 25.

ROMER

The

Vomitoxin

Story

247

*Romer, T.R.; Greaves, D. E.; Gibson, G. E. A GLC method presented at the AOAC spring workshop, Ottawa, Ontario, 1981. *Romer, T. R.; Langford, W. F. A GLC method presented at the AOAC fall meeting, Washington, D.C., 1981. Rothberg, J. M.; MacDonald, J. L.; Swims, J . C.; Romer, T. R. Proceedings of the 29th Annual Conference of Mass Spec­ trometry and Allied Topics, Minneapolis, Minnesota, May 2429, 1981. *A modification of 12. Trenholm, H. L.; Friend, D. W.; Hamilton, R. M. G.; Thomp­ son, Β. K. Publication 1745/E, available from Communica­ tions Branch, Agriculture, Canada K1A 0C7, 1982. Pollman, D. S.; Koch, B. A. Progress Report 422 - Ag. Exper. Sta., Kansas State University, Manhattan, Kansas, 66506, 1982. Forsyth, D. M.; Yoshizawa, T.; Morooka, N.; Tuite, J. Appl. and Environ. Microbiol. 1977, 34, 5, 547. Moran, E. T., Jr.; Hunter, B.; Ferket, P.; Young, L. G.; McGirr, L. G. Poultry Science 1982, 61, 1828. Cole, R.; Vesonder, R.; Lomax, L. Personal Communication. Moran, E. T . , Jr.; Hunter, R. B.; Ferket, P. R.; Young, L. G. Proceedings of the Poultry Industry School. Ontario Agricultural College, University of Guelph, 1983, 13-14. Hamilton, R. M. G. Poultry Science Annual Meeting, Univer­ sity of British Columbia, Vancouver, British Columbia, Canada, August 3-7, 1981. Trenholn, H. L. Animal Research Center, Ottawa, Ontario, Canada, Personal Communication. Mirocha, C. J.; Christensen, C. M. Ann. Rev. Phytopathology 1974, 12, 303. Cantley, T. C.; Redmer, D. Α.; Osweiler, G. D.; Day, Β. N. Fifteenth meeting Midwestern Section ASAS, March 22-24, 1982, Abstract #67. *Available from the author.

RECEIVED

June

16, 1983

16 Aflatoxins in Corn

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch016

U. L. DIENER and N. D. DAVIS Department of Botany, Plant Pathology, and Microbiology, Auburn University, Auburn, AL 36849 Certain fungi (molds) synthesize chemicals, called mycotoxins, that are poisonous and produce symptoms of toxicity when food and feed containing them is eaten by humans and animals. The aflatoxins are mycotoxins produced by two fungi, Aspergillus flavus and A. parasiticus, which are distributed worldwide in air and soils and may contaminate corn, peanuts, cottonseed, tree nuts and other crops with aflatoxin. Animal products may contain aflatoxins where contaminated feeds have been ingested. Aflatoxin B is the most potent, naturally-occurring, cancer-producing substance known, causing liver cancer in most experimental and domesticated animals and man. Preharvest infection by A. flavus may occur via colonization of corn silks followed by invasion of the glumes and surface of developing kernels. Internal seed infection occurs later during kernel denting. Commonly, ear-damaging insects transport fungal spores both externally and internally, and may provide infection sites and disperse the fungus within the ear. Stress induced by water deprivation, above normal temperatures, inadequate fertilization, and weed competition appears to exacerbate susceptibility of developing kernels to infection by A. flavus and subsequent aflatoxin biosynthesis. Aflatoxin can develop in storage; rapid drying of corn to safe storage moisture after harvest and dry storage facilities with aeration are essential to prevent contamination. Although control with fungicides and/or resistant varieties has been notably unsuccessful to date, considerable progress has been made in preventing and reducing contamination of human foods as well as losses in animal feeds by detoxifying aflatoxin-contaminated products, particularly by ammoniation. 1

0097-6156/83/0234-0249$06.25/0 © 1983 American Chemical Society

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch016

250

X E N O B I O T I C S IN

FOODS

AND

FEEDS

C e r t a i n f u n g i (molds) synthesize chemicals that are poisonous and produce symptoms of t o x i c i t y when food or feed containing them i s eaten by humans and animals. These chemicals are c a l l e d mycotoxins, a term derived from the Greek words "myces" meaning fungus and "toxikon" meaning poison. Mycotoxic e f f e c t s , such as ergot poisoning, can be traced to c i v i l i z a t i o n s of 5,000 years ago. Ergotism (St. Anthony's F i r e ) reached epidemic proportions i n the Middle Ages i n C e n t r a l Europe from the consumption of contaminated rye bread. In Russia i n the 1940s, s t a c h y b o t r y o t o x i c o s i s caused i l l n e s s and death to horses and humans, as d i d alimentary t o x i c a l e u k i a i n humans, which was a s s o c i a t e d with Fusarium i n f e c t i o n of overwintered wheat and m i l l e t ( 1 ) . The impetus that stimulated s c i e n t i f i c i n t e r e s t i n mycotoxins evolved from the death of 100,000 turkey p o u l t s at 500 l o c a t i o n s i n England i n 1960, which l e d to the discovery by B r i t i s h s c i e n t i s t s of a f l a t o x i n (a t o x i c metabolite of the fungus A s p e r g i l l u s f l a v u s Link ex F r . ) i n the peanut meal f r a c t i o n of the feed (2,3). Research soon demonstrated that a f l a t o x i n B| i s p o s s i b l y the most potent, n a t u r a l l y o c c u r r i n g carcinogen ever u t i l i z e d i n animal studies ( 4 ) . In a d d i t i o n , i t can cause acute a f l a t o x i c o s i s i n animals and humans; a case of the l a t t e r was c i t e d i n a CAST report ( 5 ) . "Perhaps the s i n g l e , most impressive, a f l a t o x i n - r e l a t e d e p i sode reported i n recent s c i e n t i f i c l i t e r a t u r e i s an acute poisoning i n an area of India i n 1974 i n v o l v i n g over 400 people and r e s u l t i n g i n 106 deaths. The circumstances were t y p i c a l of those h i g h l y conducive to excessive mycotoxin exposure, i . e . , a poor, r u r a l subsistence economy, where the people were v i r t u a l l y t o t a l l y dependent on a s i n g l e food crop (corn, Zea mays L.) that they produced themselves, and c o n d i t i o n s were favorable f o r a f l a t o x i n formation as a r e s u l t of unseasonable r a i n s that drenched the crop at harvest i n the warm s u b t r o p i c a l climate of the area. Only corn-eating e t h n i c groups and i n d i v i d u a l s were a f f e c t e d . No new cases occurred a f t e r the l o c a l l y grown corn s u p p l i e s were exhausted. Several members of some r u r a l households were a f f e c t e d . Dogs eating the same food s u f f e r e d a s i m i l a r f a t e . Medical f e a tures of the syndrome were c o n s i s t e n t with experimental data on a f l a t o x i c o s i s . Aflatoxin-contaminated corn was consumed i n a f fected households but not i n unaffected households. A f l a t o x i n concentrations were r e l a t i v e l y high with samples c o n t a i n i n g from 250 to 15,600 parts per b i l l i o n " (5). Three years l a t e r i n 1977 and again i n 1980, outbreaks of a f l a t o x i n i n corn i n Southeastern United States caused extensive crop l o s s e s and i n animals that ingested contaminated corn. This occurrence i n s t i g a t e d the development of a Regional T e c h n i c a l Committee of corn researchers i n the Southeast, which sponsored a symposium i n A t l a n t a , Georgia, i n January 1982 on " A f l a t o x i n and A s p e r g i l l u s f l a v u s i n Corn" ( 6 ) .

16.

DiENER A N D D A V I S

Aflatoxins

in Corn

251

Biology of A s p e r g i l l u s Flavus and A s p e r g i l l u s P a r a s i t i c u s

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch016

A s p e r g i l l u s f l a v u s and A. p a r a s i t i c u s Speare, c l o s e l y r e l a t e d f u n g i , occur world-wide i n the s o i l and contaminate a wide v a r i e t y of crops i n the f i e l d , during harvest, i n storage, or during p r o c e s s i n g . They are s e e d - i n h a b i t i n g f u n g i of important food crops such as corn, peanuts, cottonseed, and t r e e nuts. A f l a t o x i n s are produced only by these two f u n g i . Far l e s s i s known about the basic physiology and n u t r i t i o n of A. f l a v u s than A. p a r a s i t i c u s . Researchers have seldom d i s t i n g u i s h e d between the two species and r e f e r r e d to A. p a r a s i t i c u s as A. f l a v u s . Some morphological c h a r a c t e r i s t i c s of these two species are given i n Table I .

Table I . Morphological and Growth Comparisons of the A f l a t o x i n - p r o d u c i n g Fungi

A.

flavus

Characteristic

A.

Conidia

Distinctively verruculose

Almost smooth to l i g h t l y roughened

Conidiophore heads

Mostly u n i s e r i a t e , sometimes mixed

Consistently biseriate

Color

Ivy green

Yellow-green

Colony

Compact

Irregular

parasiticus

A. f l a v u s t y p i c a l l y produces only the t o x i c metabolites a f l a t o x i n B (AFB^) and a f l a t o x i n B (AFB ), whereas t o x i genic A. p a r a s i t i c u s i s o l a t e s produce AFG^ and AFG as w e l l as AFB^ and AFB . Over 90% of the analyses of contaminated corn samples show only AFB^ and AFB , whereas AFG^ i s common i n contaminated peanuts. A. f l a v u s i s considered dominant i n cottonseed, pecans, and other t r e e nuts as w e l l as corn. Several trace elements are r e q u i r e d f o r a f l a t o x i n formation by A. f l a v u s , p a r t i c u l a r l y z i n c ( 7 ) . The n e c e s s i t y f o r z i n c , magnesium, and other mineral elements f o r a f l a t o x i n production by A. f l a v u s (A. p a r a s i t i c u s ) i s i l l u s t r a t e d i n Table I I ( 8 ) . Z i n c , magnesium, i r o n and molybdenum were e s s e n t i a l , whereas manganese appeared to stimulate a f l a t o x i n formation. 1

2

2

2

2

2

252

XENOBIOTICS IN FOODS A N D FEEDS

Table I I .

E s s e n t i a l i t y of C e r t a i n M i n e r a l Elements to A f l a t o x i n Production by A s p e r g i l l u s f l a v u s

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch016

Element omitted

M y c e l i a l dry wt. g/100 ml

Magnesium Zinc Iron Molybdenum Copper Boron Manganese None of above A l l of above

1.60 0.60 1.33 2.86 2.85 2.91 2.12 2.77 0.65

Aflatoxin mg/100 ml B

l

0 0 0.13 0.17 0.82 0.69 1.24 0.69 0

G

l

0 0 0.16 0.30 1.76 2.00 2.64 2.00 0

Total (B+G)

0 0 0.29 0.47 2.58 2.69 3.88 2.69 0

L i l l e h o j et a l . (9) found that the a d d i t i o n of low l e v e l s of z i n c , copper, and manganese stimulated a f l a t o x i n production by A. f l a v u s growing on corn germ, as others had discovered with n u t r i e n t s o l u t i o n s (8,10). Other i n v e s t i g a t o r s have found o t h e r ­ wise, but apparently the concentration of metal s a l t determines whether i t i s stimulatory or i n h i b i t o r y (11,12), as can be noted f o r z i n c i n Table I I I (8). Thus, some of the c o n t r a d i c t i o n s i n the l i t e r a t u r e probably are due to concentration d i f f e r e n c e s i n non-comparable experiments.

Table I I I .

ZnS0 ppm

None 1 3 5 7 10

4

Influence

of Zinc L e v e l on A f l a t o x i n Production by Aspergillus flavus

M y c e l i a l dry wt. g/100 ml

0.21 2.77 2.67 3.48 3.04 3.51

Β

λ

0 0.32 0.69 0.85 0.34 0.21

Aflatoxin mg/100 ml G x

0 0.56 1.20 2.40 0.60 0.38

Total (B+G)

0 0.88 1.89 3.29 0.94 0.59

DIENER A N D DAVIS

16.

Aflatoxins

in

Corn

253

The i n f l u e n c e of v a r i o u s carbohydrates s u p p l i e d as sole carbon sources on growth and a f l a t o x i n production by A. f l a v u s , a c t u a l l y A. p a r a s i t i c u s , i s shown i n Table IV ( 8 ) . The fungus grew and produced some a f l a t o x i n on a l l of the carbohydrates t e s ­ ted except lactose* The highest y i e l d s of a f l a t o x i n were pro­ duced from glucose and sucrose.

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch016

Table IV.

Influence of Carbohydrate Source on Production of A f l a t o x i n by A s p e r g i l l u s f l a v u s

Carbohydrate (66 g/1 of carbon)

Glucose Sucrose Fructose Raffinose Mannitol Galactose Lactose Glucose + Fructose Glucose + Galactose

M y c e l i a l dry wt. g/100 ml

2.25 2.28 2.96 2.66 3.13 2.27 No Growth 2.03 2.51

Aflatoxin mg/100 ml B

l

0.76 0.76 0.38 0.42 0.03 0.02 0 0.76 0.48

G

l

1.92 1.62 0.88 0.48 0.08 0.05 0 1.84 0.93

Total (B4C)

2.68 2.38 1.26 0.90 0.11 0.07 0 2.60 1.41

The e f f e c t of sucrose concentration on a f l a t o x i n production by A. f l a v u s i s shown i n Table V (13). The highest y i e l d s were produced with 15 and 20% sucrose i n a 2% yeast e x t r a c t medium.

Table V.

Sucrose %

0 1 5 10 15 20 30 50

Influence of Sucrose Concentration on A f l a t o x i n Production by A s p e r g i l l u s f l a v u s

M y c e l i a l dry wt. g/100 ml

0.3 1.0 1.6 3.0 3.0 2.8 3.2 3.2

Β

χ

0.1 0.5 0.7 1.4 2.7 2.8 2.7 2.6

Aflatoxin mg/100 ml G

Total (B+G)

0.1 0.7 0.9 1.7 3.5 3.6 2.3 2.0

0.2 1.2 1.6 3.1 6.2 6.4 5.0 4.6

x

X E N O B I O T I C S IN F O O D S

254

AND

FEEDS

The i n f l u e n c e of n i t r o g e n source on growth and a f l a t o x i n production by A. f l a v u s (A. p a r a s i t i c u s ) i s shown i n Table VI (8). Note that the complex organic sources of n i t r o g e n , such as yeast e x t r a c t and peptone, gave tremendous y i e l d s as compared to the best s i n g l e amino a c i d . Inorganic n i t r o g e n sources, such as potassium n i t r a t e , were t o t a l l y inadequate f o r a f l a t o x i n production.

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch016

Table VI. Influence of Nitrogen Source on Growth and Production of A f l a t o x i n by A s p e r g i l l u s f l a v u s i n a Chemically Defined Medium

Nitrogen (0.42 g/1 Nitrogen)

None Yeast e x t r a c t Peptone Aspartate Glycine Glutamine Glutamate Asparagine Alanine Methionine Valine Leucine KN0 NaNo 3

3

M y c e l i a l dry g/100 ml

No Growth 3.87 2.60 3.96 4.09 3.62 3.94 3.59 3.73 3.61 3.04 2.88 3.66 3.71

Aflatoxin mg/100 ml

wt. B

l

0 2.47 1.71 0.76 0.44 0.63 0.57 0.19 0.12 0.12 0.06 0.03 0.02 0.01

G

l

0 3.20 1.56 0.64 0.74 0.53 0.58 0.18 0.15 0.09 0.05 0.03 0.02 0.01

Total (B-K?)

0 5.67 3.27 1.40 1.18 1.16 1.15 0.37 0.27 0.21 0.11 0.06 0.04 0.02

The e f f e c t of yeast e x t r a c t concentration on a f l a t o x i n production by A. f l a v u s i s shown i n Table VII (13). The highest y i e l d s were produced with 2% yeast e x t r a c t i n 20% sucrose medium. Yeast e x t r a c t provided most of the major and minor elements needed f o r growth and t o x i n production and t e c h n i c a l grade sucrose provided carbon and i r o n . Amino acids mixtures stimulate a f l a t o x i n b i o s y n t h e s i s , part i c u l a r l y asparagine and a s p a r t i c a c i d . P r o l i n e has been r e ported to stimulate c o n i d i a l germination i n a c u l t u r e that was probably A. p a r a s i t i c u s , although reported as A. f l a v u s (14). These i n v e s t i g a t o r s reported the greatest t o x i n formation with a mixture of amino acids followed by a mixture of p r o l i n e plus glutamate or p r o l i n e plus a s p a r t a t e . The e f f e c t i n the l a t t e r instances may have been due to the e f f e c t on c o n i d i a l germination rather than a d i r e c t e f f e c t on a f l a t o x i n b i o s y n t h e s i s .

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch016

16.

DiENER A N D D A V I S

Aflatoxins

in

Corn

255

The c a r d i n a l temperatures f o r vegetative growth f o r A. f l a vus are: minimum 6 to 8° C; optimum 36 to 38° C; and maximum 44 to 46° C (15). However, optima and l i m i t i n g temperatures f o r a f l a t o x i n production on media and n a t u r a l substrates vary. The optimum f o r A. f l a v u s i n l i q u i d (SMKY) medium and on peanuts was 25° C, whereas A. p a r a s i t i c u s showed highest a f l a t o x i n production at 35° C and high t o t a l a f l a t o x i n s at 30° C over i n c u b a t i o n p e r i ods of 5 to 21 days (16). P r e c i s e l i m i t i n g temperatures and r e l a t i v e humidity f o r a f l a t o x i n production oy A. p a r a s i t i c u s were determined f o r wet-heat, pasteurized peanuts, f r e s h l y - d u g peanuts and stored peanuts (17-19). L i m i t i n g temperatures were 12 and 41° C and l i m i t i n g r e l a t i v e humidity was 83% f o r sound mature k e r n e l s , broken mature (damaged) k e r n e l s , immature k e r n e l s , and kernels i n i n t a c t pods incubated f o r 84 days at 30° C. S i m i l a r data are p r e s e n t l y being obtained f o r a f l a t o x i n formation i n corn by A. f l a v u s .

Table

VII. Influence of Yeast E x t r a c t Concentration on A f l a t o x i n Production by A s p e r g i l l u s f l a v u s Growing i n 20% Sucrose Medium

Yeast e x t r a c t

%

M y c e l i a l dry wt. g/100 ml

None 0.7 2.0 3.0 5.0

0 3.3 4.1 4.2 5.2

Aflatoxin mg/100 ml B

l

0 2.4 3.6 4.3 3.0

G

l

0 3.8 4.3 3.2 2.7

Total (B-KO

0 6.2 7.9 7.5 5.7

The e f f e c t of temperature on l i n e a r growth r a t e s of A. f l a v u s and A. p a r a s i t i c u s was determined on potato-dextrose and cornmeal agars by Davis and Diener (unpublished data). A. f l a v u s grew s l i g h t l y f a s t e r than A. p a r a s i t i c u s a t 20, 27, and 35° C. Other workers measuring growth i n grams of mycelium produced reported no d i f f e r e n c e i n the two species (20,21). At best, data are meager f o r v a l i d c o n c l u s i o n s .

X E N O B I O T I C S IN

256

FOODS A N D

FEEDS

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch016

Wicklow et a l . (22) view the A. f l a v u s s c l e r o t i u m as primary inoculum i n regions where a f l a t o x i n contamination of corn i s a recurrent problem. Germination of the s c l e r o t i u m r e s u l t s i n production of c o n i d i a that represent i n f e c t i v e inoculum. D i s p e r s a l of inoculum by arthropods and wind may be f a c i l i t a t e d by s t r o n g l y roughened or echinulate c o n i d i a l w a l l s . Wicklow and Cole (23) detected aflatrem, c y c l o p i a z o n i c a c i d , and dehydroxyflavinine only i n the s c l e r o t i a from 85% of the i s o l a t e s examined and never i n the c u l t u r e medium of fungus mycelium. They argue that the s c l e r o t i u m i s important as a s u r v i v a l s t r u c t u r e and contains the chemical defense systems of the fungus as the evolutionary outcome of s e l e c t i v e f o r c e s determining the intrafungus d i s t r i b u t i o n of compounds t o x i c to p o t e n t i a l predators and p a r a s i t e s ( 6 ) . Epidemiology of A f l a t o x i n Formation by A s p e r g i l l u s Flavus Preharvest contamination of corn with a f l a t o x i n i s a serious problem. A s p e r g i l l u s f l a v u s can c o l o n i z e corn s i l k s and invade developing k e r n e l s . The most s u s c e p t i b l e stage f o r c o l o n i z a t i o n appears to be just a f t e r p o l l i n a t i o n when the s i l k s are yellowbrown i n c o l o r . At t h i s stage, A. f l a v u s c o n i d i a germinate readi l y and r a p i d l y c o l o n i z e s i l k s and p o l l e n grains on the s i l k s . In c o n t r a s t , c o n i d i a germinate poorly and l i t t l e growth occurs on u n p o l l i n a t e d s i l k s or dry brown s i l k s . Fungus i n f e c t i o n of corn s i l k s i s favored by high temperatures (Table V I I I ) (24).

Table V I I I .

Time of Inoc.S/

1 2 3 5

£./

E f f e c t of Temperature and Time of S i l k I n o c u l a t i o n on Seed I n f e c t i o n by A s p e r g i l l u s f l a v u s

Percentage of Kernels Infected at each Temperature Regime 26/22 34/22 34/30 26/30

30 46 49 14

6 4 15 3

30 5 1 1

3 6 2 1

Weeks a f t e r s i l k emergence.

In a phytotron, the percentage of i n f e c t e d kernels was 49, 15, and 2, r e s p e c t i v e l y , f o r day/night temperature regimes of 34/30, 26/30, and 26/22 C. Growth of the fungus from the e x t e r n a l s i l k s i n t o the kernels and the accompanying i n f e c t i o n

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process are not w e l l understood. The fungus can grow from e x t e r n a l s i l k s i n t o the ear i n four days. Shortly t h e r e a f t e r , the surface of the kernels and the glumes of the k e r n e l are c o l o nized. I n t e r n a l i n f e c t i o n of the k e r n e l , however, occurs l a t e r apparently during the period of k e r n e l denting. I n f e c t i o n at t h i s stage has been reported f o r other seed i n f e c t i n g f u n g i . Payne i n d i c a t e s that i n s e c t s probably play an important r o l e i n providing i n f e c t i o n s i t e s by i n j u r i n g the k e r n e l s , i n spreading the fungus with the ear, and i n bringing fungus spores i n t o the ear (6). In general, l e v e l s of contamination of i n s e c t s by A. f l a v u s ranged from 2 to 50% of the i n s e c t s observed i n preharvest corn i n c l u d i n g the corn earworm, H e l i o t h i s zea (Boddie), European corn borer, O s t r i n i a n u b i l a l i s (Hubner), and r i c e weevil, S i t o p h i l u s oryzae (L) according to M c M i l l i a n ( 6 ) . Corn-ear-inhabiting i n s e c t s have been contaminated much more with A. f l a v u s than A. p a r a s i t i c u s and the fungus has been i s o l a t e d fom a l l stages of the l i f e c y c l e of H. zea and from a l l except the egg i n S* oryzae. H. zea can also transmit A. f l a v u s from one l i f e - c y c l e stage to the next. Spore-contaminated feces have been c o l l e c t e d from H. zea l a r v a e , 24 hours a f t e r the i n g e s t i o n of A. f l a v u s c o n i d i a . The biology of s e v e r a l corn i n s e c t s i s d e t r i m e n t a l l y a f f e c t e d by A. f l a v u s , A. p a r a s i t i c u s , and/or t h e i r metabolites and they have been suggested as c o n t r o l agents. A d i r e c t causeand-effeet r e l a t i o n s h i p has been demonstrated between A. f l a v u s contaminated maize weevils (S_. zeamais Motschulsky) and A. f l a v u s contamination i n corn ears damaged by w e e v i l s . Generally, i n s e c t i c i d e a p p l i c a t i o n s and the p l a n t i n g of tight-husked, i n s e c t r e s i s t a n t hybrids have r e s u l t e d i n reduced i n s e c t damage to g r a i n and lower a f l a t o x i n l e v e l s . The c o l o n i z a t i o n of developing corn kernels by A. f l a v u s has been a s s o c i a t e d with the e a r l y milk stage (25), a f l a t o x i n has been detected i n kernels at l a t e milk stage (26), and maximum a f l a t o x i n formation has been reported when A. f l a v u s invaded k e r nels at l a t e m i l k - e a r l y dough stage (27,28). The i n t e r a c t i o n between k e r n e l moisture content (KMC) and a f l a t o x i n accumulation was studied by i n o c u l a t i o n w i t h A. f l a v u s about 20 days a f t e r s i l k i n g i n corn grown i n I l l i n o i s , M i s s o u r i , and Georgia (29). Ears were harvested 15, 30, 45, and 70 days a f t e r i n o c u l a t i o n . Data i n Table IX show that i n I l l i n o i s the KMC remained high f o r 70 days with formation of n e g l i g i b l e l e v e l s of AFB. In M i s s o u r i , AFB^ l e v e l s became s i g n i f i c a n t when KMC dropped below 30%. In Georgia, AFB l e v e l s were s i g n i f i c a n t at 51%, 31% (220 ppb), and reached 440 ppb at KMC of 14%. Some of these d i f f e r e n c e s are obviously r e l a t e d to temperature e f f e c t s r e s u l t i n g from the three d i s t i n c t r e g i o n a l l o c a t i o n s . Except under c o n t r o l l e d experiment a l c o n d i t i o n s , researchers often cannot measure d i f f e r e n c e s r e s u l t i n g from only one v a r i a b l e i n the f i e l d . X

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Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch016

Table IX.

FEEDS

Kernel Moisture Content and A f l a t o x i n B i n Corn Kernels Harvested at Various I n t e r v a l s a f t e r I n o c u l a t i o n w i t h A s p e r g i l l u s f l a v u s i n 1974 1

Post Inoc. days £/

Illinois KMC AFB^ % ppb

Missouri AFB KMC ppb %

15 30 45 70 Means

65 48 43 34 48

46 29 27 24 32

îJ

IN F O O D S A N D

1 1 5 9 4

Georgia KMC AFB % ppb

X

51 31 23 14 30

3 61 105 63 58

X

63 203 307 440 253

Inoculated 20 days a f t e r f l o w e r i n g .

Most data on the r e l a t i o n of temperature to a f l a t o x i n format i o n have been obtained with A. p a r a s i t i c u s . A f l a t o x i n contamin a t i o n of cottonseed by A. f l a v u s i n the f i e l d occurs p r i m a r i l y i n l o w - a l t i t u d e areas of Arizona and the Imperial V a l l e y of C a l i f o r n i a and not i n the hot and humid Southeastern S t a t e s . Chronic f i e l d contamination of cottonseed apparently r e q u i r e s d a i l y mean temperature of 34° C or above (T.E. R u s s e l l , personal communicat i o n ) . The s i g n i f i c a n t d i f f e r e n c e between Arizona and the Southeast c o t t o n areas i s the high night temperatures of 32-34 C i n Arizona. The r e l a t i o n of temperature to a f l a t o x i n contamination i n corn kernels from i n o c u l a t i o n at the e a r l y dough, medium dough, and l a t e dough stages of development was studied i n a phytotron (30). A f l a t o x i n l e v e l s were s i g n i f i c a n t l y higher i n kernels from p l a n t s grown at the highest combination of d a i l y temperatures (Table X). Table X.

A f l a t o x i n B^ Levels i n Corn Inoculated with A s p e r g i l l u s f l a v u s a t Three Developmental Stages and Grown under Four D i f f e r e n t P o s t i n o c u l a t i o n Temperature Regimes

Kernel Develop, at Inoc.

22/18

E a r l y Dough Medium Dough Late Dough

12.6 11.2 13.1

A f l a t o x i n Βη (ppm) Day/Night Temperature C 30/18 22/26

12.4 22.0 18.0

13.0 26.5 25.8

30/26

12.7 29.4 31.4

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259

These data and those i n Table IX emphasize the c r i t i c a l r o l e of temperature i n a f l a t o x i n formation i n developing corn k e r n e l s . Fungi are aerobic organisms and s m a l l changes i n gaseous environment can cause dramatic changes i n metabolic c e l l u l a r p r o cesses (31,32). When C 0 was removed from the atmosphere, spore germination of A. f l a v u s was i n h i b i t e d (14). In high-moisture stored corn, growth and a f l a t o x i n production by A. f l a v u s was blocked at oxygen l e v e l s below 0.5% (33). The s e n s i t i v i t y of a f l a t o x i n - p r o d u c i n g f u n g i to atmospheric gases, p a r t i c u l a r l y oxygen and carbon d i o x i d e , suggests research to determine the l e v e l of gases i n the microenvironment of developing corn k e r n e l s . Although A. f l a v u s w i l l grow on almost any n a t u r a l or processed s u b s t r a t e , a f l a t o x i n occurs n a t u r a l l y p r i m a r i l y i n corn, peanuts, cottonseed, g r a i n sorghum, tree nuts, m i l l e t , copra, and f i g s (34). Substrate f a c t o r s must be involved i n contamination, since i t i s l i m i t e d to a r e l a t i v e l y small number of a g r i c u l t u r a l commodities. The r e s t r i c t e d access of z i n c has been proposed as an explanation f o r the i n a b i l i t y of A. f l a v u s to elaborate a f l a t o x i n i n soybeans (35). The a v a i l a b i l i t y of z i n c f o r a f l a t o x i n b i o s y n t h e s i s appears to be blocked by the presence of p h y t i c a c i d i n soybeans (36). Production of a f l a t o x i n by A. f l a v u s i s r e l a t i v e l y independent of v e g e t a t i v e growth. An elevated carbon-to-nitrogen r a t i o has been l i n k e d to a f l a t o x i n formation and high t o x i n production i s dependent of high concentrations of s p e c i f i c carbohydrates i n the substrate as w e l l as s p e c i f i c organic n i t r o g e n sources. Glucose concentrations of 20-30% (37) and sucrose l e v e l s of 10-20% (13,38) have produced maximum a f l a t o x i n y i e l d s . Amino a c i d s , such as aspartate, g l y c i n e , glutamate, and glutamine, appear to promote elevated a f l a t o x i n formation (8,39). Plant s t r e s s was i n v o l v e d i n the c o r n - a f l a t o x i n outbreak i n Iowa i n 1975. That region of the s t a t e had experienced drought and above-normal temperatures, whereas other areas i n Iowa with no t o x i n contamination had normal r a i n f a l l and temperatures (40). In 1976, corn t e s t p l o t s i n s i x southern and three Corn B e l t states were sampled and monthly average temperature and r a i n f a l l were recorded (41). No a f l a t o x i n p o s i t i v e samples were taken i n Iowa, I l l i n o i s , and Ohio, which had lower temperatures than the s i x southern s t a t e s . The l o c a t i o n with the highest 3-month average temperature ( F l o r i d a ) had 75% of i t s samples a f l a t o x i n p o s i t i v e . F l o r i d a had much above normal r a i n f a l l i n June (+4.6") and much below normal i n J u l y (-3.4) and August (-5.4). Thus, d i s t i n c t drought s t r e s s was present i n these l a s t two months. Other research has been summarized (42) by a proposal that elevated temperatures and water s t r e s s are r e s p o n s i b l e f o r i n creased p a r a s i t i c a b i l i t y of A. f l a v u s , a s s o c i a t e d i n f e c t i o n processes, and higher a f l a t o x i n c o n c e n t r a t i o n i n developing k e r n e l s (Table XI). 2

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A N D FEEDS

Table XI. E f f e c t of Water Stress on Kernel I n f e c t i o n and A f l a t o x i n Production by A s p e r g i l l u s f l a v u s

Treatment

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch016

No Stress P r e i n o c u l a t i o n Stress Postinoculation Stress

Infected kernels %

40 38 35

Aflatoxin l PPb B

28 291 392

Jones et a l . (43) a l s o found a f l a t o x i n was l e s s severe i n the high c o r n - y i e l d i n g Tidewater region than i n the sandy C o a s t a l Plain soils. Stress was l i n k e d to reduced water h o l d i n g c a p a c i t y of the s o i l and drought r e l a t e d i n t e r f e r e n c e with n u t r i e n t uptake by corn p l a n t s . A c o n t r o l l e d i r r i g a t i o n study during 1977 and 1978 showed 23.6% a f l a t o x i n - p o s i t i v e samples (7.3 ppb AFB^) from i r r i g a t e d p l o t s as compared to 54.9% p o s i t i v e samples (61.9 ppb AFB^) from n o n - i r r i g a t e d p l o t s . This convincing evidence of an a s s o c i a t i o n between water s t r e s s on developing corn p l a n t s and enchanced v u l n e r a b i l i t y of the kernels to a f l a t o x i n contamin a t i o n p a r a l l e l e d the f i n d i n g s of P e t t i t et a l . (44) i n 1971 on the e f f e c t of i r r i g a t i o n on a f l a t o x i n accumulation i n peanuts. F e r t i l i t y s t r e s s from reduced f e r t i l i z a t i o n and dense popul a t i o n s of plants c o n t r i b u t e s to a f l a t o x i n development i n p r e harvest corn (26). A d e f i n i t e r e l a t i o n s h i p between increased a f l a t o x i n l e v e l s i n kernels from p l a n t s grown under n i t r o g e n s t r e s s has been reported (45). The competition of weeds f o r a v a i l a b l e n u t r i e n t s and water a l s o has been l i n k e d to presence of a f l a t o x i n i n corn (46). Sampling and A n a l y s i s In aflatoxin-contaminated l o t s of corn, a small percentage of k e r n e l s may contain very high concentrations of a f l a t o x i n making accurate determinations of a f l a t o x i n l e v e l s d i f f i c u l t ( 6 ) . This f a c t o r makes g e t t i n g a r e p r e s e n t a t i v e sample f o r chemical a n a l y s i s the c r i t i c a l step. Research i n d i c a t e s that the variance and skewness of the d i s t r i b u t i o n of a f l a t o x i n concentrations of samples about a l o t mean are i n v e r s e l y p r o p o r t i o n a l to sample s i z e and to t o x i n c o n c e n t r a t i o n i n the l o t . The d i s t r i b u t i o n of sample concentrations about the l o t mean i s p r a c t i c a l l y normal when l o t c o n c e n t r a t i o n i s greater than 20 ppb a f l a t o x i n and sample weight i s 4.45 kg or g r e a t e r . Probe sampling may be used when a l o t of corn has been r e c e n t l y blended i n h a r v e s t i n g , handl i n g , o r other operations; otherwise, stream sampling should be used.

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A number of r e l i a b l e e f f e c t i v e methods of d e t e c t i o n and determination of a f l a t o x i n i n corn are a v a i l a b l e (6)· (1) the b r i g h t greenish-yellow fluorescence (BGYF) presumpt i v e t e s t or black l i g h t t e s t i s widely used by farmers, e l e v a t o r operators, and i n d u s t r y to i d e n t i f y suspect l o t s of corn that may contain a f l a t o x i n . It alone does not demonstrate the presence of a f l a t o x i n , but requires a chemical a n a l y s i s f o r confirmation. (2) For l a b o r a t o r i e s with minimal equipment and requirements f o r r e s u l t s i n a minimum amount of time, the Holaday-Velasco minicolumn method has been adopted f o r a f l a t o x i n i n corn by the A s s o c i a t i o n of O f f i c i a l A n a l y t i c a l Chemists (AOAC) as w e l l as the American A s s o c i a t i o n of C e r e a l Chemists (AACC). (3) The o f f i c i a l method i s by the CB method, but an improved method i s needed that i s l e s s expensive and uses l e s s t o x i c s o l vents. Modified and a l t e r n a t e methods abound but are untested except by t h e i r c r e a t o r . There are s e v e r a l promising q u a n t i t a t i v e methods that need e v a l u a t i o n i n c o l l a b o r a t i v e s t u d i e s by the AOAC and the AACC. High performance l i q u i d chromatography (HPLC), the f l u o r o m e t r i c i o d i n e method (FL-IM), the enzyme-linked immunosorbent assay (ELISA), the solid-phase radioimmunoassay (RIA), and the tandem mass spectrometer, a l l have great p o t e n t i a l . Research i s needed to determine the extent, l i m i t a t i o n s , and a p p l i c a t i o n s f o r each method. B i o l o g i c a l E f f e c t s of A f l a t o x i n i n Domestic Animals Bovine. Although acute a f l a t o x i c o s i s i n c a t t l e r e s u l t s i n overt symptoms, such as reduced feed consumption, severely depressed m i l k - y i e l d , weight l o s s , and l i v e r damage, the i n s i d i o u s b i o l o g i c a l e f f e c t s r e s u l t i n g from chronic exposure to sub-acute l e v e l s of a f l a t o x i n may be of greater economic importance ( 6 ) . Low to moderate intakes of aflatoxin-contaminated r a t i o n s by d a i r y and beef c a t t l e have been a s s o c i a t e d with reduced feed e f f i c i e n c y , immunosuppression, increased s u s c e p t i b i l i t y to s t r e s s , and reduced reproductive performance. These low l e v e l e f f e c t s are d i f f i c u l t to recognize; thus, the incidence of chronic a f l a t o x i c o s i s i n c a t t l e as w e l l as other l i v e s t o c k may be greater than reported. Not only i s animal h e a l t h a f f e c t e d during chronic a f l a t o x i c o s i s , but a l s o a p o r t i o n of ingested AFB^ i s converted to a hydroxylated metabolite, AFM^, and excreted i n milk. Because the conversion r a t e i s approximately 0.9%, a d a i r y cow may produce milk containing some AFM^ without manifesting symptoms of i n t o x i c a t i o n . Research concerning the b i o l o g i c a l e f f e c t s of AFBi i n the bovine has been p r i n c i p a l l y designed f o r t e s t i n g acute t o x i c o l o g i c a l response. L i t t l e d e f i n i t i v e data are a v a i l able on the e f f e c t s of a f l a t o x i n on p h y s i o l o g i c a l processes, such as n u t r i e n t ( p a r t i c u l a r l y t r a c e mineral) metabolism or c e l l mediated immune response. Porcine. Data from v e t e r i n a r y d i a g n o s t i c l a b o r a t o r i e s

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i n d i c a t e that porcine a f l a t o x i c o s i s was common i n 1981 i n South­ eastern and Midwestern United States (6). Twenty to 60% of corn and feed samples suspected of t o x i c i t y contained a f l a t o x i n and concentrations exceeding 200 ppb were common. Suckling p i g l e t s , growing and f i n i s h i n g swine, and breeding stock are s u s c e p t i b l e to a f l a t o x i c o s i s , but c l i n i c a l and p a t h o l o g i c a l a l t e r a t i o n s vary g r e a t l y depending upon age, d i e t , dosage, and duration of ex­ posure. Decrease i n the rate of weight gain and impaired feed conversion e f f i c i e n c y are among the mildest e f f e c t s , and l e t h a l , acute, severe, t o x i c h e p a t i t i s , nephrosis, and systemic hemor­ rhages are among the most severe. A f l a t o x i n s are carcinogenic i n p i g s , but toxin-induced n e o p l a s i a i n pigs have not been recog­ nized to occur n a t u r a l l y . Spontaneous porcine a f l a t o x i c o s i s i s a s s o c i a t e d with increased s u s c e p t i b i l i t y to disease and e x p e r i ­ mental a f l a t o x i c o s i s may cause immunosuppression i n pigs ( 6 ) . The conditions under which t h i s occurs however are incompletely de­ f i n e d . A f l a t o x i n s are absorbed r a p i d l y from the porcine gas­ t r o i n t e s t i n a l t r a c t and are concentrated l a r g e l y i n the l i v e r and kidney. E x c r e t i o n occurs mostly i n the feces and i n l e s s e r amounts i n the u r i n e . A f l a t o x i n s B^, B , G^, G , M^, M and a f l a t o x i c o l are metabolites now i d e n t i f i e d i n p i g s . Avian. A f l a t o x i n (AFB^) from peanut meal caused s i g n i f i ­ cant losses of turkeys, ducklings and pheasants i n England, 1960 (2,3). Research demonstrated that exposure to AFB^ r e s u l t e d i n hepatic c e l l damage with n e c r o s i s , congestion, and b i l e duct pro­ l i f e r a t i o n (2). There were macroscopic hemorrhages, i c t e r u s , and the l i v e r s were f i r m and pale. Large, f o c a l areas of lymphoid h y p e r p l a s i a and m i t o t i c f i g u r e s were observed. A f l a t o x i n i s r e ­ cognized as an important carcinogen. A f l a t o x i n (0.25-0.5 yg/g B^) consumed during periods of immunization against P a s t e u r e l l a multocida i n t e r f e r e d with the development of acquired r e s i s t a n c e i n 20-67% of turkey poults and chicks (47). Decreased immunity p e r s i s t e d even when an a f l a t o x i n - f r e e d i e t was s u b s t i t u t e d . This immunosuppression r e s u l t e d from impairment of the r e t i c u l o e n d o ­ t h e l i a l system. A f l a t o x i n B^ (0.2 yg/g) i n commercial feed f o r New Hampshire and b r o i l e r chicks decreased weight gains, and increased s u s c e p t i b i l i t y and m o r t a l i t y from c e c a l c o c c i d i o s i s (48) . In another t r i a l , vaccinated and nonvaccinated groups of chickens exposed to AFB^ were more s u s c e p t i b l e to challenge i n o c u l a t i o n with Marek s v i r u s than s i m i l a r AFB^ f r e e groups (49) . In f u r t h e r t r i a l s i n turkey poults (50), i n c l u s i o n of AFB^ i n the d i e t at 0.5 yg/g reduced leukocyte counts as w e l l as serum complement t i t e r s . A l s o , serum γ - g l o b u l i n s increased during the AFB^ exposure p e r i o d . Toxic e f f e c t s were diminished by the a d d i t i o n of 2 yg/g Selenium (Se) i n the d i e t (50). S e l e ­ nium, at concentrations that increased l i v e r g l u t a t h i o n e p e r o x i ­ dase (GSH.Px) a c t v i t y , probably d e t o x i f i e d the AFBj^-2,3 oxide to reduced GSH. Phagocytic a c t i v i t y was markedly suppressed i n 2

2

2

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the AFB^ exposed groups; t h i s suppression of a c t i v i t y was prevented i n poults exposed to AFB^ and r e c e i v i n g d i e t a r y Se at 2.0 yg/g. H e p a t o t o x i c i t y i n turkeys poults from AFB^ exposure probably r e s u l t e d i n Se d e f i c i e n c y , reducing b i l e flow with diminished absorption of vitamin A r e s u l t i n g i n increased membrane p e r m e a b i l i t y , edema, and hemorrhage. Equine. C l i n i c a l signs and symptoms of equine a f l a t o x i c o s i s are v a r i a b l e and do not o f f e r a c l e a r pathognomonic p i c t u r e of the disease ( 6 ) . Of the hematologic values examined, prolonged c l o t t i n g time appeared to be s i g n i f i c a n t . E l e v a t i o n s of bloodbiochemical enzymes, such as glutamic o x a l o a c e t i c transaminase, l a c t a t e dehydrogenase, i d i t o l dehydrogenase, gamma glutamyl t r a n s f e r a s e , and arginase during the t o x i c i t i e s were reported. L i v e r n e c r o s i s and hemorrhagic l e s i o n s were found i n most of the animals necropsied along with h i s t o p a t h o l o g i c damage to the l i v e r , kidney, heart, and b r a i n . Toxic l e v e l s of a f l a t o x i n r e ported i n c l i n i c a l cases ranged from 54.8 ppb to 6,500 ppb. A f l a t o x i n B^ and M^ were i s o l a t e d from feed, f e c e s , and l i v e r tissues. Economic E f f e c t s of A f l a t o x i n i n Corn Corn i s important to the g r a i n and l i v e s t o c k economy of the Southeast. In recent years, g r a i n i n f e c t i o n and a f l a t o x i n contamination have become s e r i o u s problems and unless these problems are solved, f u t u r e growth of the g r a i n and l i v e s t o c k i n d u s t r i e s i n the r e g i o n could be adversely a f f e c t e d ( 6 ) . Losses from a f l a t o x i n occur at a l l l e v e l s i n the production, marketing, and u t i l i z a t i o n process. Contaminated g r a i n causes s e r i o u s marketing problems f o r producers and g r a i n buyers. Some e l e v a t o r s refuse to buy corn i f i t t e s t s p o s i t i v e above FDA a c t i o n g u i d e l i n e s of 20 ppb. Other buyers may purchase corn at higher l e v e l s of a f l a t o x i n , but discount the p r i c e paid according to the amount of a f l a t o x i n i n the sample analyzed. L i v e s t o c k and p o u l t r y producers have become acutely aware of the p o t e n t i a l problem r e l a t e d to contaminated feeds. Many problems with animal performance, disease, and death have been a t t r i b u t e d to aflatoxin-contaminated feed, although i t has f r e q u e n t l y been impossible to determine the a c t u a l cause of the problems. Nearly 30 l a w s u i t s , t o t a l i n g over $8 m i l l i o n i n damages, have been f i l e d against g r a i n and feed firms by l i v e s t o c k feeders i n the Southeastern States during the past three years. A d d i t i o n a l costs r e s u l t i n g from a f l a t o x i n contamination of corn include (a) s u r v e i l l a n c e and assays by s t a t e departments of a g r i c u l t u r e and (b) l e g i s l a t i v e appropriations f o r research and extension e f f o r t s . Summary of l o s s e s are shown i n Table XII t o t a l i n g over $400 m i l l i o n f o r the two epidemic years of 1977 and 1980.

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264

Table XII.

FEEDS

Cost of A f l a t o x i n i n Corn i n Southeast

Data i n $1,000

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FOODS A N D

P r i v a t e cost: *J Corn producer Handler Swine Milk Poultry Cornmeal Public costs: S u r v e i l l a n c e and assay Research and extension TOTAL

£/ Milk and cornmeal estimate ^ Data from NC & GA i n 1977;

1977

79, 939 7, 592 58, 691

— 50, 000

— 545 800 197, 567

1980

97, 157 13, 997 100, 360 2 25, 000 145 673 500 237, 834

from NC o n l y . NC only i n 1980.

While the short-run costs are s u b s t a n t i a l f o r the i n d i v i d u a l and f o r s o c i e t y , they may be greater i n the long run i f r e current a f l a t o x i n contamination i n corn cannot be eliminated or d e t o x i f i e d . Farmers, who are unable to market the corn, w i l l u l timately s h i f t acreage to other crops, which have l e s s y e a r - t o year r i s k s and considerably l e s s net r e t u r n s . Fewer buyers may be needed to handle the crops that are produced and marketed. Some feeders, p a r t i c u l a r l y p o u l t r y i n t e g r a t o r s , are already purchasing Midwestern g r a i n i n preference to l o c a l g r a i n . Others are c o n s i d e r i n g b r i n g i n g g r a i n from the Midwest i n u n i t t r a i n s to lower t h e i r c o s t s . Absence of corn production i n the Southeast could a l t e r some of the comparative advantage of the area i n feeding c e r t a i n s p e c i e s , such as b r o i l e r s and hogs. However, some of the e f f e c t on p o u l t r y producers may be dampened by f u r ther downward adjustments i n g r a i n f r e i g h t rates from the Midwest. These changes could impact adversely on both producers and consumers. Prevention and C o n t r o l of A f l a t o x i n i n Corn Screening Methods. Screening to detect r e s i s t a n c e i n corn to k e r n e l i n f e c t i o n by A. f l a v u s and/or a f l a t o x i n production have been conducted i n the f i e l d and to a l e s s e r extent i n the l a b o r a tory (6). To date, screening f o r genotypic d i f f e r e n c e s has focused p r i m a r i l y on l e v e l of a f l a t o x i n production rather than on frequency of k e r n e l i n f e c t i o n . I n o c u l a t i o n methods have g e n e r a l l y included some form of wounding of the kernels to e s t a b l i s h the

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fungus i n ears, although recent studies show that i n f e c t i o n and subsequent a f l a t o x i n production can occur i n the absence of wounding. In screening f o r r e s i s t a n c e to the fungus or t o x i n production, researchers are f a c i n g problems that are more f o r midable than those u s u a l l y encountered. Future progress i n screening w i l l be f a c i l i t a t e d by: 1) i d e n t i f i c a t i o n of at l e a s t one r e s i s t a n t check; 2) improvements i n uniformity of treatment response among r e p l i c a t i o n s , l o c a t i o n , and years; 3) development of i n o c u l a t i o n methods that y i e l d i n f e c t i o n l e v e l s of s u f f i c i e n t magnitude to d i f f e r e n t i a t e among genotypes; and 4) development of a r a p i d and inexpensive method f o r a f l a t o x i n assessment. I d e n t i f i c a t i o n of plant characters (morphological or biochemical) that reduce i n f e c t i o n or a f l a t o x i n synthesis may provide a s i g n i f i c a n t step forward. Plant Breeding. D i f f e r e n c e s i n a f l a t o x i n l e v e l s i n preharvest commercial corn hybrids have been reported i n i s o l a t e d i n stances (6). However, i n a b i l i t y to repeat d i f f e r e n c e s among hyb r i d s over l o c a t i o n s and years has been a major obstacle to recommending one hybrid over another f o r reducing a f l a t o x i n contamination. Studies have shown that maize parents can be c l a s s i f i e d as donors of low, intermediate, and high a f l a t o x i n product i o n to t h e i r r e s p e c t i v e progenies. These r e s u l t s may i n d i c a t e that a f l a t o x i n contamination i s under genetic c o n t r o l , but d i f ferences among genotypes have been so e r r a t i c that recurrent s e l e c t i o n i s not c u r r e n t l y warranted. In genetic s t u d i e s , a large i n t e r a c t i o n between genotype and environment has been observed; t h i s i n t e r a c t i o n probably has masked d i f f e r e n c e s i n a f l a t o x i n a s s o c i a t e d with i n h e r i t e d response. Many f a c t o r s may c o n t r i b u t e to the large e r r o r s encountered i n c l u d i n g : sampling technique, a f l a t o x i n a n a l y s i s , i n o c u l a t i o n method, number of samples, a lack of knowledge of the host-pathogen r e l a t i o n s h i p , and varying environmental c o n d i t i o n s . E l u c i d a t i o n of the chemic a l basis f o r genetic c o n t r o l of the i n f e c t i o n and contamination process would probably help reduce the large experimental e r r o r s . G e n e t i c a l l y c o n t r o l l e d , i n d i r e c t methods of reducing preharvest a f l a t o x i n contamination i n c l u d e p l a n t i n g hybrids with adaptation to l o c a t i o n and a high degree of r e s i s t a n c e to ear-damaging i n s e c t s , diseases, and s t r e s s during the g r a i n f i l l i n g period. In a d d i t i o n , the choice of 1) p l a n t i n g date, 2) optimum plant dens i t y , and 3) good c u l t u r a l p r a c t i c e s , such as weed c o n t r o l and f e r t i l i t y balance to a l l e v i a t e s t r e s s during the g r a i n f i l l i n g period, should be u s e f u l i n c o n t r o l l i n g the a f l a t o x i n problem. Natural M e t a b o l i t e s . An innovative area of research i s the e f f e c t of corn metabolites on A. f l a v u s , A. p a r a s i t i c u s , and a f l a t o x i n production. Metabolites from s e v e r a l p l a n t s have been reported to i n h i b i t A. f l a v u s or a f l a t o x i n s y n t h e s i s . A number of chemicals c o n s i s t i n g of v o l a t i l e o i l s from corn husks, k e r n e l s , and s i l k s were extracted by s e v e r a l i n v e s t i g a t o r s (51,52) and tested i n c u l t u r e by Wilson et a l . (53). Several a l c o h o l s

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and aldehydes were i n h i b i t o r y to A. f l a v u s with β-ionone dramati­ c a l l y r e s t r i c t i n g growth, s p o r u l a t i o n , and a f l a t o x i n production by A. f l a v u s and A. p a r a s i t i c u s . N a t u r a l l y o c c u r r i n g compounds may be u s e f u l i n plant breeding o r i n chemical c o n t r o l of A. f l a v u s i n the f i e l d or i n storage* P e s t i c i d e C o n t r o l . Numerous a n t i m i c r o b i a l compounds have been studied i n the l a s t 10 to 15 years seeking means of c o n t r o l ­ l i n g growth of A s p e r g i l l u s f l a v u s and a f l a t o x i n production i n corn ( 6 ) . A number of chemicals, i n c l u d i n g p e s t i c i d e s , i n h i b i t a f l a t o x i n production i n the l a b o r a t o r y , but only the propionates and some acetates have been found e f f e c t i v e i n stored corn. Over 30 p e s t i c i d e s have been tested f o r t h e i r a b i l i t y to i n h i b i t growth and a f l a t o x i n production by A. f l a v u s , but only f i v e have been a p p l i e d to corn during the growing season to determine i f preharvest development of a f l a t o x i n can be reduced or prevented. The i n s e c t i c i d e Gardona when sprayed three times per week r e ­ duced, but d i d not e l i m i n a t e a f l a t o x i n B^ accumulation on pre­ harvest corn (54). Preharvest corn that was n a t u r a l l y contamina­ ted by A. f l a v u s a f t e r k e r n e l damage contained 28.9 ppb a f l a t o x i n B^. A p p l i c a t i o n of the i n s e c t i c i d e s bux, c a r b a r y l , and dyfonate reduced a f l a t o x i n %i l e v e l s to 4 ppb, 1 ppb, and 2 ppb, r e ­ s p e c t i v e l y (55). D e t o x i f i c a t i o n . Outbreaks of a f l a t o x i n i n corn occur spo­ r a d i c a l l y i n d i f f e r e n t sections of the United S t a t e s . In recent years, incidence has centered l a r g e l y i n the Southeastern S t a t e s . When these outbreaks occur, some means of salvaging the contami­ nated corn must be considered. Numerous approaches have been suggested, l a r g e l y f a l l i n g i n the broad categories of p h y s i c a l separation, chemical i n a c t i v a t i o n , and b i o l o g i c a l i n a c t i v a t i o n . Research conducted on peanut and cottonseed meals has provided a b a s i s f o r the s e l e c t i o n of methods to be a p p l i e d to the d e t o x i f i ­ c a t i o n of corn. A review of the l i t e r a t u r e i n d i c a t e s that the most p r a c t i c a l method f o r salvaging aflatoxin-contaminated corn i s ammoniation. Extensive work a t the Northern Regional Research Center r e s u l t e d i n development of a procedure whereby i n t r o d u c ­ t i o n of ammonia reduced a f l a t o x i n i n corn from i n excess of 1,000 ppb to l e s s than 10 ppb (56,57,58). Although t o x i c o l o g i c a l s t u ­ dies have not yet been completed to q u a l i f y the process f o r FDA approval, i n d i c a t i o n s are that the method i s f e a s i b l e and o f f e r s promise to farmers and buyers, who may otherwise face f i n a n c i a l d i s a s t e r as a r e s u l t of a f l a t o x i n contamination of corn.

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XENOBIOTICS IN FOODS A N D F E E D S

Lillehoj, E. B.; Kwolek, W. F.; Vandegraft, Ε. E.; Zuber, M. S.; Calvert, O. H.; Widstrom, N.; Futrell, M. C.; Bockholt, A. C. Crop Sci. 1975, 15, 267-270. Thompson, D. L . ; Lillehoj, Ε. B.; Leonard, K. J.; Kwolek, W. F.; Zuber, M. S. Crop Sci. 1980, 20, 609-612. Detroy, R. W.; Lillehoj, Ε. B.; Ciegler, A. "Microbial Toxins"; Ciegler, Α.; Kadis, S.; Ajl, S. J., Eds.; Academic: New York, 1971, Vol. VI, p. 3-178. Shih, C. N.; Marth, Ε. H. Biochem. Biophys. Acta 1974, 338, 286-296. Wilson, D. M.; Jay, E. Appl. Microbiol. 1975, 29, 224-228. Stoloff, L. "Mycotoxins in Human and Animal Health"; Rodricks, J. V.; Hesseltine, C. W.; Mehlman, Μ. Α., Eds.; Pathotox: Park Forest South, Ill., 1977, p. 7-28. Gupta, S. K.; Venkitasubramanian, T. A. Z. Lebensm. Unters. Forsch. 1975, 159, 107-111. Gupta, S. K.; Venkitasubramanian, T. A. Appl. Microbiol. 1975, 29, 834-836. Shih, C. N.; Marth, Ε. H. Appl. Microbiol. 1974, 27, 452-456. Llewellyn, G. C.; Jones, H. C.; Gates, J. E . ; Eadie, T. J. Assoc. Off. Anal. Chem. 1980, 63, 622-625. Mateles, R. I.; Adye, J. C. Appl. Microbiol. 1965, 13, 208-211. Lillehoj, Ε. B.; Hesseltine, C. W. "Mycotoxins in Human and Animal Health"; Rodricks, J. V.; Hesseltine, C. W.; Mehlman, Μ. Α., Eds.; Pathotox: Park Forest South, Ill., 1977, p. 107-119. Lillehoj, Ε. B.; Kwolek, W. F.; Zuber, M. S.; Calvert, O. H.; Horner, E. S.; Widstrom, N. W.; Guthrie, W. D.; Scott, G. E.; Thompson, D. L.; Findley, W. R.; Bockholt, A. J. Cereal Chem. 1978, 55, 1007-1013. Jones, R. K.; Duncan, Η. E.; Payne, G. Α.; Leonard, H. J . Plant Dis. 1980, 64, 859-863. Jones, R. K.; Duncan, Η. E.; Hamilton, P. B. Phytopathology 1981, 71, 810-816. Pettit, R. E.; Taber, R. Α.; Schroeder, H. W.; Harrison, A. L. Appl. Microbiol. 1971, 22, 629-634. Jones, R. K.; Duncan, Η. E. Plant Dis. 1981, 65, 741-744. Cobb, W. Y. Quart. Bull. Assoc. Food Drug Off. 1979, 43, 99-107. Pier, A. C.; Heddleston, K. L. Avian Dis. 1970, 14, 797-809. Edds, G. T.; Simpson, C. F. Am. J . Vet. Res. 1976, 37, 65-68. 49. Edds, G. T.; Nair, K. P. C.; Simpson, C. F. Am. J. Vet. Res., 1973, 34, 819-826. Goldstein, S. L. M.S. Thesis, University of Florida, Gainesville, 1981. Buttery, R. G.; Ling, L. C.; Chan, B. G. J. Agric. Food Chem. 1978, 26, 866-869.

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch016

16.

DIENER A N D DAVIS

Aflatoxins

in

Corn

52. Flath, R. Α.; Forrey, R. R.; John, J. O.; Chan, B. G. J. Agric. Food Chem. 1978, 26, 1290-1293. 53. Wilson, D. M.; Gueldner, R. C.; McKinney, J. M.; Livesay, R. H.; Evans, B. D.; Hill, R. A. J. Am. Oil Chem. Soc. 1981, 58, 959A-961A. 54. Widstrom, N. W.; Lillehoj, Ε. B.; Sparks, A. N.; Kwolek, W. F. J. Econ. Entomol. 1976, 69, 677-679. 55. Elahi, M.; Draughon, F. A. Proc. 41st Inst. Food Technol. Mtg. 1981, p. 111. 56. Brekke, O. L.; Peplinski, A. J.; Lancaster, Ε. B. Trans. ASAE 1977, 20, 1160-1168. 57. Brekke, O. L.; Peplinski, A. J.; Nofsinger, G. W.; Conway, H. F.; Stringfellow, A. C.; Montgomery, R. R.; Silman, R. W.; Sohns, V. E . ; Bagley, Ε. B. Trans. ASAE 1979, 22, 425-432. 58. Brekke, O. L . ; Stringfellow, A. C.; Peplinski, A. J. J. Agric. Food Chem. 1978, 26, 1383-1389. RECEIVED June

6, 1983

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17 Detection of Trichothecene Mycotoxins Quantitation of Deoxynivalenol by Negative Chemical Ionization Mass Spectrometry JANET M. ROTHBERG, JOHN L. MACDONALD, and JOSEPH C. SWIMS

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch017

Central Research Services, Ralston Purina Company, St. Louis, MO 63164

A GC/MS technique has been developed for the detection of trichothecene mycotoxins. The technique has been used to quantitate the trichothecene deoxynivalenol in corn, wheat and mixed feeds. The trichothecenes are derivatized with heptafluorobutyrylimidazole and the derivatives are separated by gas chromatography. Methane negative chemical ionization produces characteristic fragment ions with high molecular weights which are measured by selected ion monitoring. The technique has been used to determine low levels of six trichothecene compounds: diacetoxyscirpenol, neosolaniol, T-2 toxin, HT-2 toxin, deoxynivalenol, and fusarenon-x. One hundred femtograms of deoxynivalenol has been detected by this method. The coefficients of variation of the method for quantitation of deoxynivalenol in both corn and wheat are 15% with a mean recovery of greater than95%in spiked corn and wheat. Levels of deoxynivalenol which were measured by different laboratories using a variety of methods will be compared. Trichothecences are a class o f s t r u c t u r a l l y similar m y c o t o x i n s p r o d u c e d p r i n c i p a l l y by F u s a r i u m m o l d s . T h e s e c y c l i c compounds a r e o f i n t e r e s t t o f e e d m a n u f a c t u r e r s b e c a u s e t h e y c a n c a u s e f e e d r e f u s a l o r r e d u c e d f e e d e f f i c i e n c i e s i n some a n i m a l s p e c i e s ( 1 , 2 , 3 ) . S e v e r a l a p p r o a c h e s have been r e p o r t e d f o r t h e analysis o f trichothecenes i n feeds and feed i n g r e d i e n t s . T r i m e t h y l s i l y l d e r i v a t i v e s o f t h e t r i c h o t h e c e n e s have been formed and t h e d e r i v a t i v e s measured by g a s c h r o m a t o g r a p h y u s i n g a f l a m e i o n i z a t i o n d e t e c t o r (4,5,6,7). Other workers obtained improved 0097-6156/83/0234-0271$06.00/0 © 1983 A m e r i c a n C h e m i c a l S o c i e t y

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s e n s i t i v i t y by f o r m i n g t h e h e p t a f l u o r o b u t y r y l (HFB) d e r i v a t i v e s o f t h e t r i c h o t h e c e n e s w h i c h c o u l d t h e n be measured by e l e c t r o n c a p t u r e gas c h r o m a t o g r a p h y ( 5 , 8 , 9 ) . The use o f mass s p e c t r o m e t r y has a l s o been r e p o r t e d f o r c o n f i r m a t i o n and q u a n t i t a t i o n o f t h e t r i c h o t h e c e n e s d e o x y n i v a l e n o l (DON), d i a c e t o x y s c i r p e n o l (DAS), T-2 t o x i n and HT-2 t o x i n ( 1 0 , 1 1 , 1 2 ) . The t e c h n i q u e d e s c r i b e d h e r e u s e s gas c h r o m a t o g r a p h y combined w i t h n e g a t i v e c h e m i c a l i o n i z a t i o n (NCI) mass spectrometry to analyze e x t r a c t s o f feed i n g r e d i e n t s . H e p t a f l u o r o b u t y r y l d e r i v a t i v e s o f t h e t r i c h o t h e c e n e s a r e formed by r e a c t i o n w i t h h e p t a f l u o r o b u t y r y l i m i d a z o l e (HFBI) and t h e d e r i v a t i v e s a r e t h e n s e p a r a t e d by g a s - l i q u i d c h r o m a t o g r a p h y . C h a r a c t e r i s t i c h i g h m o l e c u l a r w e i g h t (542-943 amu) n e g a t i v e i o n s a r e measured u s i n g s e l e c t e d i o n m o n i t o r i n g ( S I M ) . The methods l i s t e d above g e n e r a l l y r e q u i r e some t r e a t m e n t o f t h e sample e x t r a c t s p r i o r t o gas c h r o m a t o g r a p h i c a n a l y s i s t o remove substances which i n t e r f e r e with q u a n t i t a t i o n o f the t r i c h o t h e c e n e s . The c o m b i n a t i o n o f s e n s i t i v i t y and s e l e c t i v i t y r e s u l t i n g from t h e use o f NCI and s e l e c t e d i o n m o n i t o r i n g p e r m i t s a n a l y s i s o f sample e x t r a c t s w i t h o u t a d d i t i o n a l sample c l e a n - u p . D e t e c t i o n o f s i x t r i c h o t h e c e n e s ; DON, DAS, T-2 t o x i n , HT-2 t o x i n , n e o s o l a n i o l ( N S L ) , f u s a r e n o n e - x (F-X) i s shown h e r e . T h i s method has been u s e d f o r q u a n t i t a t i o n o f DON i n c o r n , wheat, wheat b r a n , and mixed f e e d s . The p r e c i s i o n o f t h e method was measured by r e p e t i t i v e a n a l y s i s o f n a t u r a l l y c o n t a m i n a t e d s a m p l e s . R e c o v e r y was m e a s u r e d from samples s p i k e d w i t h DON standards. Experimental D e o x y n i v a l e n o l s t a n d a r d was p u r c h a s e d from M y c o l a b s , I n c . , Chesterfield, Missouri. Additional deoxynivalenol, d i a c e t o x y s c i r p e n o l and T-2 t o x i n were p u r i f i e d f r o m F u s a r i u m c u l t u r e s i n t h i s l a b o r a t o r y . The HT-2 t o x i n s t a n d a r d was o b t a i n e d from Sigma C h e m i c a l Company, S t . L o u i s , MO. F u s a r e n o n - x and n e o s o l a n i o l were p r o v i d e d by D r . L o s h i o Ueno, T o k y o U n i v e r s i t y o f S c i e n c e , Tokyo, J a p a n . The n - h e p t a f l u o r o b u t y r y l i m i d a z o l e r e a g e n t was o b t a i n e d from R e g i s C h e m i c a l Company, M o r t o n Grove, I l l i n o i s . S o l v e n t s were o f a q u a l i t y s u i t a b l e f o r p e s t i c i d e a n a l y s i s and were o b t a i n e d f r o m various sources. A l l mass s p e c t r a were r e c o r d e d u s i n g a H e w l e t t - P a c k a r d 5985B mass s p e c t r o m e t e r e q u i p p e d w i t h a d u a l e l e c t r o n i m p a c t / c h e m i c a l i o n i z a t i o n s o u r c e i n c l u d i n g n e g a t i v e i o n c a p a b i l i t y . The s o u r c e t e m p e r a t u r e was 125°C, f i l a m e n t e m i s s i o n c u r r e n t was 300 uA, and e l e c t r o n e n e r g y was 230 e v . The i n t e r f a c e between t h e GC and MS was a g l a s s - l i n e d s t a i n l e s s s t e e l t r a n s f e r l i n e . Methane was used b o t h f o r c a r r i e r gas and a s t h e c h e m i c a l i o n i z a t i o n r e a c t a n t g a s . A methane f l o w o f 11.5 mL/min. r e s u l t e d i n a n i o n s o u r c e p r e s s u r e o f 0.5 t o r r .

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Samples were a n a l y z e d o n a 6 f t χ 2 mm ID g l a s s c o l u m n p a c k e d w i t h 3% SP-1000 ( S u p e l c o , B e l l e f o n t e , P A . ) . The column t e m p e r a t u r e was programmed from 150°C t o 225°C a t 1 0 ° C / m i n . The i n j e c t i o n p o r t t e m p e r a t u r e was 2 0 0 ° C and t h e i n t e r f a c e t e m p e r a t u r e was 2 5 0 ° C . The i n s t r u m e n t was o p e r a t e d i n t h e s e l e c t e d i o n m o n i t o r i n g (SIM) mode. T h i s a l l o w e d g r o u p s o f one t o f o u r i o n s t o b e m o n i t o r e d a t a t i m e . M a s s e s t o be m o n i t o r e d were changed p r i o r to the r e t e n t i o n time o f each t r i c h o t h e c e n e d e r i v a t i v e .

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch017

Samples C o r n c o n t a m i n a t e d w i t h DON was o b t a i n e d from t h e 1980 c o r n c r o p from E a s t e r n Canada. The c o r n was s e l e c t e d f o r a n a l y s i s due t o t h e p r e s e n c e o f mold o n t h e k e r n e l s . Wheat c o n t a m i n a t e d w i t h DON was t a k e n from samples o f t h e 1980 wheat c r o p from t h e upper m i d w e s t e r n U n i t e d S t a t e s . Wheat s a m p l e s were s e l e c t e d f o r a n a l y s i s due t o t h e p i n k c o l o r o f t h e g r a i n . Samples o f n a t u r a l l y c o n t a m i n a t e d c o r n and wheat were b l e n d e d t o p r o d u c e samples c o n t a i n i n g v a r i o u s l e v e l s o f DON. F o l l o w i n g g r i n d i n g , samples c o n t a i n i n g h i g h l e v e l s o f DON were b l e n d e d w i t h samples which had been a s s a y e d by t h e method d e s c r i b e d h e r e and f o u n d t o c o n t a i n low l e v e l s o f DON. These b l e n d e d samples were u s e d t o measure t h e p r e c i s i o n o f t h e method and p r o v i d e c o n t r o l s a m p l e s t o be a n a l y z e d w i t h e a c h s e t o f unknown s a m p l e s . A r t i f i c i a l l y DON c o n t a m i n a t e d s a m p l e s were p r e p a r e d by a d d i n g a l i q u o t s from s t a n d a r d s o l u t i o n s o f DON i n a c e t o n e t o g r o u n d samples o f c o r n and wheat. A f t e r a d d i t i o n , t h e s o l v e n t was a l l o w e d t o e v a p o r a t e b e f o r e a n a l y s i s . These samples were u s e d t o measure r e c o v e r y o f DON t h r o u g h t h e method. Procedure E x t r a c t i o n . A 25-g g r o u n d sample was e x t r a c t e d i n 100 mL o f 50% m e t h a n o l / w a t e r ( v / v ) . The m i x t u r e was b l e n d e d a t t h e h i g h e s t s p e e d o f a n e x p l o s i o n - p r o o f b l e n d e r . The sample was t h e n c e n t r i f u g e d a t 28,000 rpm. f o r 5 m i n u t e s . Sample e x t r a c t s were r e f r i g e r a t e d o v e r n i g h t i f t h e y c o u l d n o t b e a n a l y z e d o n t h e same day. D e r i v a t i z a t i o n . A 1 mL a l i q u o t f r o m t h e c e n t r i f u g e d sample e x t r a c t was d i l u t e d t o 200 mLs i n a v o l u m e t r i c f l a s k . A 1 mL a l i q u o t o f t h i s d i l u t e d e x t r a c t was t r a n s f e r r e d t o a s m a l l s c r e w - c a p p e d c u l t u r e t u b e w h i c h was t h e n p l a c e d i n a 50°C w a t e r b a t h and d r i e d u n d e r a s t r e a m o f n i t r o g e n . Then t h e sample was t h e n d i s s o l v e d i n 1 mL o f 95% t o l u e n e / 5 % a c e t o n i t r i l e ( v / v ) a n d mixed v i g o r o u s l y f o r a t l e a s t 30 s e c o n d s . Two h u n d r e d m i c r o l i t e r s o f n - h e p t a f l o u r o b u t r y l i m i d a z o l e (HFBI) was added t o e a c h sample and t h e s o l u t i o n was a g a i n mixed v i g o r o u s l y f o r 15 seconds.

X E N O B I O T I C S IN F O O D S A N D

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274

FEEDS

E x c e p t f o r t h o s e samples a n d s t a n d a r d s c o n t a i n i n g DAS, t h e samples were p l a c e d i n a 1 0 0 ° C o i l b a t h f o r 15 m i n u t e s . A f t e r r e m o v a l o f t h e sample f r o m t h e o i l b a t h , 3 mL o f 5% sodium b i c a r b o n a t e s o l u t i o n was a d d e d t o e a c h sample a n d t h e n m i x e d v i g o r o u s l y f o r 30 s e c o n d s . T h e sample was a l l o w e d t o phase, t h e o r g a n i c l a y e r was removed a n d t r a n s f e r r e d t o a c u l t u r e t u b e c o n t a i n i n g 300 mg o f a n h y d r o u s sodium s u l f a t e c r y s t a l s . T h i s t u b e was t h e n s h a k e n b r i e f l y . F i v e m i c r o l i t e r s o f t h e d e r i v a t i z e d sample was i n j e c t e d i n t o t h e GC/MS. The d i a c e t o x y s c i r p e n o l (DAS) s t a n d a r d a n d s a m p l e s c o n t a i n i n g DAS were d e r i v a t i z e d a t room t e m p e r a t u r e f o r 15 m i n u t e s . A t 1 0 0 ° C l o s s e s o f DAS have been e x p e r i e n c e d . GC/MS A n a l y s i s . The s i x t r i c h o t h e c e n e s were d e t e c t e d by m o n i t o r i n g t h e m o l e c u l a r i o n (M+l) a n d c h a r a c t e r i s t i c f r a g m e n t i o n s o f e a c h d e r i v a t i v e . The i o n s m o n i t o r e d a r e shown i n T a b l e I I . Q u a n t i t a t i o n o f t h e d e o x y n i v a l e n o l d e r i v a t i v e (DON-HFB) were 670, 671, 884, a n d 885 amu. Results and D i s c u s s i o n Confirmation of Trichothenes The b a s i c s k e l e t a l s t r u c t u r e o f t h e F u s a r i u m t r i c h o t h e c e n e s i s shown i n F i g u r e I . The s i x t r i c h o t h e c e n e s d i s c u s s e d h e r e d i f f e r i n t h e number a n d p o s i t i o n o f h y d r o x y l g r o u p s , a c e t a t e e s t e r s , o r i s o b u t y l e s t e r s s u b s t i t u t e d a t one o f f o u r s i t e s o n t h e m o l e c u l e . When t h e t r i c h o t h e c e n e i s d e r i v a t i z e d w i t h HFBI, h e p t a f l u o r o b u t r y r l e s t e r s a r e formed w i t h h y d r o x y l g r o u p s o n t h e t r i c h o t h e c e n e m o l e c u l e . D e p e n d i n g o n t h e number o f h y d r o x y l g r o u p s , 1,2,or 3 HFB g r o u p s may be added, t h u s i n c r e a s i n g t h e m o l e c u l a r w e i g h t o f t h e compound by 196 amu f o r e a c h HFB group a t t a c h e d . The r e s u l t i n g d e r i v a t i v e s have m o l e c u l a r w e i g h t s i n t h e r a n g e o f 540-950 amu. The NCI s p e c t r a a r e g e n e r a l l y c h a r a c t e r i z e d by a n a b u n d a n t m o l e c u l a r i o n , t h e l o s s o f h y d r o g e n f l u o r i d e (M-20) g r o u p s , a n d l o s s o f i n t a c t HFB (M-213) g r o u p s . T a b l e I shows t h e t a b u l a t e d NCI s p e c t r a o f s i x d e r i v a t i z e d t r i c h o t h e c e n e s . F o r example, i n t h e c a s e o f d e o x y n i v a l e n o l - H F B , t h e m o l e c u l a r i o n i s m/e= 884, l o s s o f HF(M-20) r e s u l t s i n m/e= 864, l o s s o f i n t a c t HFB g r o u p s (M-213) g i v e s f r a g m e n t i o n s o f m/e= 671,458. T a b l e I . T a b u l a t e d NCI S p e c t r a o f S i x T r i c h o t h e c e n e D e r i v a t i v e s Compound Deoxynivalenol/tri-HFB Fusarenon-x/tri-HFB Neosolaniol/di-HFB HT-2/di-HFB Diacetoxyscirpenol/HFB T-2/HFB

884 942 692 816 480 580

Mass I n t e n s i t y ( 6 6 ) , 671 ( 5 ) , 6 7 0 ( 7 ) , 6 3 0 ( 2 ) ( 1 0 0 ) , 9 4 3 ( 3 5 ) , 728 ( 9 ) , 922 ( 5 ) ( 9 7 ) , 754 ( 5 1 ) , 774 ( 2 5 ) , 693 ( 2 4 ) ( 1 0 0 ) , 817 ( 3 6 ) , 233 ( 2 2 ) , 583 ( 6 ) ( 1 0 0 ) , 481 ( 2 6 ) , 562 ( 2 ) , 542 ( 9 ) ( 1 0 0 ) , 581 ( 3 0 ) , 6 6 2 ( 4 ) , 642 ( 8 )

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One o f t h e a d v a n t a g e s o f t h i s GC/MS method o f d e t e c t i o n o f the d e r i v a t i z e d trichothecenes i s the reduction of the e f f e c t o f t h e i n t e r f e r e n c e s from t h e sample m a t r i x . Many i n t e r f e r e n c e s a r e s m a l l m o l e c u l a r w e i g h t compounds, but t h e l a r g e m o l e c u l a r w e i g h t t r i c h o t h e c e n e d e r i v a t i v e s c a n be s e l e c t i v e l y m o n i t o r e d a t t h e h i g h e r mass i o n s . I n t e r f e r e n c e s s e e n i n t h e e l e c t r o n c a p t u r e c h r o m a t o g r a p h i c d e t e r m i n a t i o n o f t r i c h o t h e c e n e s due t o t h e e x c e s s HFBI l e f t i n t h e e x t r a c t a f t e r d e r i v a t i z a t i o n and due t o t h e low m o l e c u l a r w e i g h t compounds f r o m t h e sample m a t r i x a r e n o t a p p a r e n t i n NCI mass s p e c t r o m e t r y . A second advantage i s t h a t attachment o f the e l e c t r o n e g a t i v e HFB g r o u p s t o t h e t r i c h o t h e c e n e s r e s u l t s i n v e r y h i g h s e n s i t i v i t y when u s i n g n e g a t i v e c h e m i c a l i o n i z a t i o n T h i s s e n s i t i v i t y i s d e m o n s t r a t e d by t h e d e t e c t i o n o f a 300 femtogram i n j e c t i o n o f DON-HFB a s shown i n F i g u r e I I . A c o m p a r i s o n o f n e g a t i v e c h e m i c a l i o n i z a t i o n (NCI), p o s i t i v e chemical i o n i z a t i o n (PCI), and e l e c t r o n i m p a c t ( E I ) s e n s i t i v i t i e s t o d e r i v a t i z e d DON s t a n d a r d i s shown i n T a b l e I I . E a c h s o u r c e t e c h n i q u e was r u n o n t h e same i n s t r u m e n t o n t h e same day. The NCI t e c h n i q u e i s a b o u t 5000 t i m e s more s e n s i t i v e t h a n PCI, and 10,000 t i m e s more s e n s i t i v e than E I . C o n f i r m a t i o n o f t r i c h o t h e c e n e contaminated samples i s p e r f o r m e d by s e l e c t e d i o n m o n i t o r i n g (SIM) mass s p e c t r o m e t r y , scanning the molecular i o n of each t r i c h o t h e c e n e d e r i v a t i v e . U s i n g SIM, c o e l u t i n g p e a k s c a n be s e p a r a t e d and q u a n t i t a t e d w i t h o u t d i f f i c u l t y . F i g u r e I I I shows s e p a r a t i o n o f s i x t r i c h o t h e c e n e s t a n d a r d s t h a t were d e r i v a t i z e d s e p a r a t e l y and c o m b i n e d b e f o r e i n j e c t i o n . The m o l e c u l a r i o n was m o n i t o r e d f o r e a c h compound i n t h i s d e m o n s t r a t i o n . E v e n t h o u g h , under t h e c o n d i t i o n s u s e d h e r e , NSL and DAS a r e n o t c o m p l e t e l y r e s o l v e d , t h e y c a n e a s i l y be d e t e c t e d i n d e p e n d e n t l y w i t h SIM. T a b l e I I . L i m i t o f D e t e c t i o n O f T h r e e Mass S p e c t r a l Techniques to Deoxynivalenol Standard Technique E l e c t r o n Impact P o s i t i v e Chemical I o n i z a t i o n Negative Chemical I o n i z a t i o n

Lowest DON S t a n d a r d M e a s u r e a b l e 100 p g 500 p g 0.1 p g

Quantitation of Deoxynivalenol The method has been u s e d f o r t h e q u a n t i t a t i o n o f d e o x y n i v a l e n o l i n c o r n , wheat, and m i x e d f e e d s . The p r e c i s i o n and r e c o v e r y o f t h e method have been e s t a b l i s h e d i n o u r l a b o r a t o r y . The p r e c i s o n s t u d y was p e r f o r m e d o n l a r g e b a t c h e s o f n a t u r a l l y c o n t a m i n a t e d c o r n and wheat. C o r n c o n t a i n i n g low l e v e l o f DON was g r o u n d , m i x e d , and r i f f l e d w i t h h i g h D O N - l e v e l c o r n t o g i v e a l a r g e homogeneous s a m p l e o f c o n t a m i n a t e d c o r n w i t h a l e v e l o f a p p r o x i m a t e l y 1500 ppb.

XENOBIOTICS

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch017

276

Figure

Figure

R2

R3

R4

one ORc ORc OH ORc hi

ORc ORc ORc ORc OH OH

H H H H OH OH

H OOCBu OH OOCBu - 0 - 0

of six trichothecenes

2. NCI spectrum

of DON

FEEDS

COMPOUND

Rl

1. Structure

INFOODS A N D

DIACETOXYSCIRPENOL T-2 TOXIN NEOSOLANIOL HT-2 TOXIN FUSRRENON-X DETOXYN1VRLENOL

showing four groups for

standard (100 femtograms) sensitivity.

showing

substitutions.

instrument

ROTHBERG ET AL.

Detection

of Trichothecene

I

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch017

-I

j

^

m'Z 8 1 6 Μ

.ut.i

β

7

6

277

Mycotoxins

8

9

m/z

662

IB

Figure 3. Separation of six combined derivatives of trichothecene standards selected ion monitoring. A series of unique ion fragments are monitored analysis. (Molecular ions only are shown in figure.)

m- z

DON

884

HT-2 m ·-' ζ

816

12

Figure

4. Confirmation

of DON

and HT-2 in naturally

13

contaminated

14

corn.

by for

X E N O B I O T I C S IN F O O D S A N D

278

FEEDS

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch017

The same p r e p a r a t i o n was p e r f o r m e d w i t h wheat. A t l e a s t e i g h t p o r t i o n s o f e a c h o f t h e c o r n and wheat samples were e x t r a c t e d , d i l u t e d , d e r i v a t i z e d , and a n a l y z e d by mass s p e c t r o m e t r y . Q u a n t i t a t i o n o f t h e DON i n t h e sample m a t r i c e s was a c c o m p l i s h e d by measurement o f t h e peak a r e a o f t h e m o l e c u l a r i o n o f t h e DON-HFB i n t h e sample and c o m p a r i n g t h i s t o t h e peak a r e a o f t h e same i o n a s t h e s t a n d a r d . T a b l e I I I shows p r e c i s i o n d a t a o n t h e r e p l i c a t e samples o f c o r n and wheat. The c o e f f i c i e n t o f v a r i a t i o n o f t h e v a l u e s f o r t h e a r t i f i c a l l y c o n t a m i n a t e d c o r n was l e s s t h a n 11%. E i g h t samples o f t h e a r t i f i c i a l l y c o n t a m i n a t e d wheat samples were r u n , t h e CV was 10%. T a b l e I I I . P r e c i s i o n D a t a o n Wheat and C o r n A r t i f i c i a l l y Contaminated with Deoxynivalenol Ν Corn Wheat

Mean

11 8

1456 1466

PPB DON Range

CV

1187-1682 1 1 . 4 % 1245-1720 1 0 . 3 %

T a b l e IV shows t h e r e c o v e r y d a t a f o r DON-spiked m a t r i c e s o f b o t h c o r n and wheat. The s p i k e l e v e l was v a r i e d t o c o v e r t h e range o f v a l u e s c o r r e s p o n d i n g t o t h e l e v e l s f o u n d i n t h e n a t u r a l l y - c o n t a m i n a t e d m a t r i c e s . The mean r e c o v e r y f o r b o t h c o r n and wheat samples was 97%. T a b l e IV. CORN:

WHEAT:

R e c o v e r i e s i n DON-Spiked M a t r i c e s D a t a

Spike Level 170 ppb 200 860 1000 2000

Ν Average Recovery 3 126% 3 87% 13 105% 2 78% 3 85% Mean R e c o v e r y f o r a l l c o r n s p i k e s = Spike Level Ν Average Recovery 1000 ppb 3 94% 2000 ppb 3 100% Mean R e c o v e r y f o r a l l wheat samples

C.V. 11% 17% 19% 33% 25% 97% C.V. 11% 8% = 97%

To compare r e s u l t s f o r q u a n t i t a t i o n o f DON by v a r i o u s methods, samples o f c o n t a m i n a t e d c o r n , wheat and m i x e d f e e d s were s u b m i t t e d t o s i x l a b s who u s e d v a r i o u s methods f o r q u a n t i t a t i o n o f DON. The methods o f a n a l y s i s i n c l u d e d gas c h r o m a t o g r a p h y / e l e c t r o n c a p t u r e (GC/EC) method, (6) a c o m b i n e d

17.

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of Trichothecene

279

Mycotoxins

t h i n - l a y e r c h r o m a t o g r a p h y ( T L C ) , GC, and MS a n a l y s i s , (17) R o m e r s GC/EC method, (15) and t h e method d e s c r i b e d i n t h i s p a p e r . The r e s u l t s o f t h e c o r r e l a t i o n s t u d y a r e shown i n T a b l e V. The s t u d y shows t h a t t h e r e i s c o n s i d e r a b l e v a r i a t i o n between l a b o r a t o r i e s , r e g a r d l e s s o f t h e method o f a n a l y s i s . The r e s u l t s o f t h e a n a l y s i s by t h e NCI-mass s p e c t r o m e t r i c method d e s c r i b e d i n t h i s p a p e r , a r e g e n e r a l l y s l i g h t l y h i g h e r t h a n o b t a i n e d by o t h e r methods. One p o s s i b l e e x p l a n a t i o n f o r t h e h i g h e r r e s u l t s o b t a i n e d w i t h t h i s method may be h i g h e r r e c o v e r i e s o b t a i n e d by NCI-MS compared t o r e c o v e r i e s r e p o r t e d f o r o t h e r p r o c e d u r e s . T a b l e V I shows a c o r r e l a t i o n s t u d y between two l a b o r a t o r i e s o n s a m p l e s o f wheat b r a n a n a l y z e d f o r d e o x y n i v a l e n o l . The agreement between t h e two l a b o r a t o r i e s was good, t h e r e s u l t s o b t a i n e d by S c o t t ' s method (6) a g r e e d w e l l w i t h t h e r e s u l t s o b t a i n e d by t h e NCI-MS t e c h n i q u e .

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch017

1

T a b l e V: D e o x y n i v a l e n o l C o r r e l a t i o n S t u d y

Sample Corn Corn Wheat Wheat Wheat Mixed Mixed Mixed

Lab A 1280 40 1310 1250 200 F e e d 200 F e e d 4960 F e e d 7100

DON i n Lab Β 1000 500 1000 1000 500 1000 500 500

ppb Lab C 1000 10 1090 1150 150 * * *

Lab D 600 300 800 800 300 300 4800 6400

Lab Ε 1710 200 630 830 200 200 4080 5180

Lab F 760 70 990 830 160 130 2920 4500

Lab G 1580 20 1380 1510 174 380 7410 10000

Method

(5)

(17)

(5)

(11)

(15)

(1)

NCIMass S p e c .

%CV Mean ** 1160 ~38 130 20 1030 28 1062 27 240 13 240 54 4110 40 6640 32

* Lab c h o s e n o t t o r u n m i x e d f e e d s . ** E x c l u d e s Lab Β r e s u l t s . V a l u e s r e p o r t e d a s " l e s s t h a n " a r e n o t i n c l u d e d i n mean/CV. T a b l e V I : Wheat B r a n C o r r e l a t i o n S t u d y D e o x y n i v a l e n o l Sample Wheat B r a n Wheat B r a n Wheat B r a n Wheat B r a n Wheat C o n t r o l Method

Level(ppb)

Lab A 627 504 514 728 201

Lab Β 620 497 600 754 312

NCI Mass Spec

(5)

280

X E N O B I O T I C S IN F O O D S A N D

FEEDS

Conclusion N e g a t i v e i o n c h e m i c a l i o n i z a t i o n mass s p e c t r o m e t r y c a n be u s e d t o c o n f i r m t r i c h o t h e c e n e s a n d q u a n t i t a t e DON i n n a t u r a l l y c o n t a m i n a t e d g r a i n s a n d f e e d s . The e f f e c t s o f i n t e r f e r e n c e s a r e e l i m i n a t e d by s e l e c t i v e l y s c a n n i n g t h e r e l a t i v e l y h i g h m o l e c u l a r weight fragments and molecular i o n o f t h e t r i c h o t h e c e n e d e r i v a t i v e s . Separation o f a mixture o f trichothecene d e r i v a t i v e s i s a c c o m p l i s h e d i n 15 m i n u t e s o n a p a c k e d GC c o l u m n . Acknowledgment The a u t h o r s a c k n o w l e d g e t h e a s s i s t a n c e o f Thomas R. Romer, o f R o m e r s L a b , I n c . , f o r t h e e a r l y work done o n g a s c h r o m a t o g r a p h y separation o f the derivatized trichothecenes, f o r h i s assistance i n t h e p r o c u r e m e n t o f s a m p l e s a n d s t a n d a r d s . Acknowledgment i s a l s o g i v e n t o Dave G r e a v e s , f o r m e r l y o f R a l s t o n P u r i n a Company f o r t h e e x t r a c t i o n o f t h e samples, and t o Roland Laramore, R a l s t o n P u r i n a Co., a n d F r a n O l i v i g n i , f o r m e r l y o f R a l s t o n P u r i n a Company, f o r t h e p r e p a r a t i o n o f t h e d e o x y n i v a l e n o l , d i a c e t o x y s c i r p e n o l , a n d t h e T-2 t o x i n s t a n d a r d s .

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch017

f

Literature Cited 1. Vesonder, R. F.; Ciegler, Α.; Jensen, A. H. Appl. Microbiol. 1973, 26, 1008-1010. 2. Forsyth, D. M.; Yoshizawa, T.; Mooroka, N.; Tuite, J. Appl. Environ. Microbiol 1977, 34, 547-552. 3. Vesonder, R. F.; Ciegler, Α.; Burmeister, H. R.; Jensen, A. H. Appl. Environ. Microbiol. 1979, 38,344-346. 4. Ikediobi, C. O., Hus, I. C.; Bamburg, J. R; Strong, F. M. Anal. Biochem. 1979, 43, 327-340. 5. Scott, P. M., Lau, P-4; Kanhere, S. R. Meeting of Assoc. of Official Analytical Chemists 1981. 6. Pareless, S. R.; Collins, G. J.; Rosen, J. D. J. Agric. Food Chem. 1976, 24 (4), 872-875. 7. Ikediobi, C.O., Hsu, I. C.; Bamburg, J. R.; Strong, F. M. Anal Biochem. 1970, 43, 327-340. 8. Szathmary, Cs.; Galacz, J.; Vida, L.; Alexander, G. J. Chrom. 1980, 191, 327-331. 9. Ueno, Y.; Sat, N.; Ishii, K.; Sakai, K.; Tsunoda, H.; Enomoto M. Appl. Micro 1973, 25 (4), 699-704. 10. Romer, T. R.; Boling, T. M.; MacDonald, J. L. J. Assoc. Off. Anal Chem. 1978, 61, 801-808. 11. Romer, T. R.; Greaves, D. Α.; Langford, W. L. 95th Annu. Mtg Assoc. Off. Anal. Chem., October 19-22, 1981, Washington, D. C. 12. Ishii, J., Y. Ando, and Ueno, Y. Chem. Pharm Bull 1975, 23 (9), 2152-2164.

17.

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of Trichothecene

Mycotoxins

281

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch017

13. Rothberg, J. M.; MacDonald, J. L.; Swims, J. C.; Romer T. R. American Soc. for Mass Spec. May, 1981, Minneapolis. "Analysis of Trichothecences Using Chemical Ionization Mass Spectroscopy." 14. MacDonald, J. L.; Romer, T. R. Proc 25th Annual Conf. Mass Spec. Allied Topics, 1977. Washington, D.C. 15. Romer, T. R.; Greaves, D. Α.; Gibson, G. I. Assoc. Off. Analytical Chemists, May, 1981, Ottawa, Ontario, Canada. 16. Romer, T. R.; Boling, T. M. 93rd Annu. Mtg. Assoc. Off. Anal. Chem., October 15-18, 1979, Washington, D. C. 17. Stahr, H. M., Lerdal, D., Hyde, W., Pfeiffer, R. Amer. Assn. Veterinary Lab Diagnosticians 1981, 24th Ann. Proceedings, 277-286. RECEIVED

July 15, 1983

18 Antinutrients and Allergens in Oilseeds ROBERT L. ORY, ANTONIO A. SEKUL, and ROBERT R. MOD

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch018

Southern Regional Research Center, U.S. Department of Agriculture, ARS, New Orleans, LA 70179 The presence of antinutrients and allergens in foods and feeds has prompted much research on these xenobiotics as concern for the safety of foods. Some of the more prominant adventitious components of peanuts and soybeans (allergens, trypsin inhibitors, hemagglutinins), cottonseed (allergens, gossypol, cyclopropene fatty acids), and castor seed (allergens, toxins), their effects on selected enzymes and metabolism of mammals, on the general well-being of humans, some of the chemical/biochemical methods used to identify these materials, their subcellular site in the seeds, and methods for measuring them are described. A n t i n u t r i e n t s and a l l e r g e n s have always been present i n c e r t a i n foods but the growing i n t e r e s t i n o i l s e e d s as a source of e d i b l e p r o t e i n as well as o i l , and current i n t e r e s t i n xenobiot i c s and safety of foods i s focusing more a t t e n t i o n on the a n t i n u t r i e n t s , such as t r y p s i n i n h i b i t o r s , hemagglutinins, gpssypol, toxins, e t c . I n 1979, however, Dr. P h i l i p L. White, D i r e c t o r of the Department of Food and N u t r i t i o n f o r the American Medical A s s o c i a t i o n , s a i d , "Nothing has happened i n the l a s t 1, 5, 10, or 50 years that has suddenly made our food supply hazardous. I f anything i t i s much less hazardous today than i t was 75 years ago" (I). Despite such statements, people today are not sure what foods are safe or which ones c o n t a i n a l l e r g e n s , a n t i n u t r i ents, carcinogens, toxins, e t c . Consumers are a l s o paying more a t t e n t i o n to nutrient contents of both f r e s h and prepared foods. To keep pace with t h i s awareness of n u t r i t i o n a l content by consumers, research has increased on the safety, wholesomeness, and s t a b i l i t y of both t r a d i t i o n a l foods and new food products entering the market, e s p e c i a l l y those from plant sources. T h i s report

T h i s chapter not subject to U . S . copyright. P u b l i s h e d 1983, A m e r i c a n C h e m i c a l S o c i e t y

284

X E N O B I O T I C S IN F O O D S A N D

d e s c r i b e s a few of t h e s e a n t i n u t r i e n t s , e m p h a s i z i n g t h e i r c h e m i s t r y and the a n a l y t i c a l methods u s e d t o measure them than t h e i r p h y s i o l o g i c a l a c t i v i t y i n humans.

FEEDS

rather

Food A l l e r g i e s . The terra " a l l e r g i e " was i n t r o d u c e d b y v o n P i r q u e t (2) i n 1906 t o d e s c r i b e t h e change i n a p e r s o n s c a p a c i t y to r e a c t to a second i n j e c t i o n of h o r s e serum. Allergens are g e n e r a l l y large m o l e c u l a r weight m a t e r i a l s too large to d i a l y z e out of s o l u t i o n and are m o s t l y g l y c o p r o t e i n i n n a t u r e . Allerg e n i c a c t i v i t y i s most o f t e n a s s o c i a t e d w i t h the p r o t e i n m o i e t y of the compound. The most f r e q u e n t f o o d s t h a t i n d u c e a l l e r g i c r e a c t i o n s i n h u m a n s a r e c o w ' s m i l k , c h o c o l a t e , b e v e r a g e s made f r o m t h e K o l a n u t , c o r n , e g g s , t h e pea f a m i l y of legumes (mostly p e a n u t s , w h i c h a r e not nuts but a r e l e g u m e s ) , c i t r u s , tomatoes, wheat and o t h e r s m a l l g r a i n s , cinnamon and a r t i f i c i a l food c o l o r s (3). E x t r a c t s of p e a n u t s f r e q u e n t l y c r o s s r e a c t w i t h a n t i b o d i e s to v a r i o u s beans and peas, e s p e c i a l l y soybeans. Cereal grains i n c l u d e many s t a p l e f o o d s p r e p a r e d f r o m w h e a t , s u c h a s b r e a d a n d p a s t a p r o d u c t s , some w h i c h h a v e b e e n r e p o r t e d t o p r o d u c e a l l e r g i c r e a c t i o n s i n h u m a n s . Wheat i s p r o b a b l y t h e most f r e q u e n t offender and wheat g l u t e n h y p e r s e n s i t i v i t y , c a l l e d c e l i a c d i s e a s e , i n d u c e s a d v e r s e r e a c t i o n s i n humans s e n s i t i v e t o wheat g l u t e n .

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch018

1

I n c l i n i c a l d i a g n o s i s of s e n s i t i v i t y t o a l l e r g e n s i n f o o d , t h e two m e t h o d s u s e d most f r e q u e n t l y a r e t h e s k i n p r i c k t e s t (SPT) and the r a d i o a l l e r g o s o r b e n t t e s t (RAST). These t e s t s have been u s e d r e l i a b l y f o r the d i a g n o s i s of a l l e r g y to peas, c o d f i s h , p e a n u t s , egg w h i t e , wheat and wheat f l o u r b u t were o n l y p a r t l y r e l i a b l e i n d e t e c t i n g a l l e r g y to cow's m i l k , s a r d i n e s , and white b e a n s ( 4_) . Oilseed Allergens. Legumes and o i l s e e d s c o n t a i n l a r g e amounts of storage and n o n - s t o r a g e p r o t e i n s . Most storage p r o t e i n s are g l o b u l i n s s o l u b l e i n d i l u t e s a l t or b u f f e r and i n s o l u b l e i n water. The n o n - s t o r a g e p r o t e i n s c o n t a i n w a t e r - s o l u b l e a l b u m i n s , enzymes and g l y c o p r o t e i n s t h a t i n c l u d e t r y p s i n i n h i b i t o r s , hema g g l u t i n i n s , a l l e r g e n s , and o t h e r x e n o b i o t i c s which a f f e c t nutrit i o n a l v a l u e of the p r o t e i n . Many of t h e s e a r e o f t e n a s s o c i a t e d with storage proteins. Some a l l e r g e n s f o u n d i n o i l s e e d s a n d t r e e n u t s a r e shown i n T a b l e 1. Allergen concentrations i n oilseeds a p p e a r t o be much h i g h e r t h a n t h o s e i n t r e e n u t s , p e a n u t s a n d soybeans (two legumes). Except for castor beans, cottonseeds, and f l a x s e e d , the y i e l d s of r e c o v e r e d a l l e r g e n s a r e generally l e s s t h a n 1%. N i t r o g e n c o n t e n t s of these a l l e r g e n s range from 11% t o 18% b u t c a r b o h y d r a t e c o n t e n t s s h o w m u c h w i d e r v a r i a t i o n : 3% t o 3 9 % . P e a n u t s a p p e a r t o be more a l l e r g e n i c t h a n t r e e n u t s , perhaps because they a r e consumed i n l a r g e r q u a n t i t i e s . Amino a c i d p r o f i l e s of the a l l e r g e n s i n c o t t o n s e e d s , castor s e e d s , a n d p e a n u t s show more s i m i l a r i t y i n t h e f o r m e r two s e e d s t h a n w i t h t h e p e a n u t 05). C a s t o r seed and c o t t o n s e e d a l l e r g e n s

18.

ORY E T AL.

Table I .

Antinutrients

and Allergens

in Oilseeds

285

Some A l l e r g e n s i n Tree Nuts, O i l s e e d s , and Legumes

%

%

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch018

Seed

Allergen*

Yield

% Ν

Carb.

Almonds B r a z i l Nuts Filberts

IA IA IA

0.46 0.81 0.30

16.9 17.6 17.1

12 .6 6.9 10.5

Castor Seeds Cottonseed Flaxseed

IA IA IA

1.76 1.38 1.98

18.4 12.1 11.3

3.1 36.4 39.4

Peanuts

IA IB IB

0.07 0.18 0.10

15.4 15.2 13.3

16.7 10.6 20.4

Soybeans

* I s o l a t e d by method of Spies, et a l . (5); % N=nitrogen; % Ca r b. - ca rb ohy dra t e.

have high a r g i n i n e and glutamic a c i d concentrations (lower i n peanuts), and they have low l y s i n e (higher i n peanut a l l e r g e n ) . Glycine content i n the peanut a l l e r g e n , PN-1B, i s 5-10 times higher than that i n castor and cottonseed a l l e r g e n s , CB-1A and CS-1A. Neither amino a c i d s , percent n i t r o g e n , or percent carbohy­ drate contents are consistent enough to be c o r r e l a t e d with allergenic sensitivity. Peanuts. Peanuts are one of the world's major o i l s e e d s . They have been reported to contain various compounds such as hemag­ g l u t i n i n s (6), antiheraophilia f a c t o r s (7), t r y p s i n i n h i b i t o r s ( 8 ) , and a l l e r g e n s (5). Spies et ajL (5) were the f i r s t to chemi­ c a l l y c h a r a c t e r i z e i s o l a t e d peanut a l l e r g e n s , but when they d i d t h i s research, peanut a l l e r g e n s were not considered a hazard. Recently s e v e r a l reports of a l l e r g i c r e a c t i o n to peanuts by humans have appeared (9,10). Reports i n the l i t e r a t u r e today concern a l l e r g e n s present i n raw peanuts. However, people who show p o s i t i v e r e a c t i o n s to pea­ nut a l l e r g e n s are g e n e r a l l y a l l e r g i c to roasted peanut products. By Spies' (5) c l a s s i f i c a t i o n , PN-1A i s heat-stable whereas PN-1B i s h e a t - s e n s i t i v e and would be destroyed i n the r o a s t i n g process. PN-1A i s present i n extremely low amounts i n the seed. We examin­ ed raw and roasted peanut a l l e r g e n e x t r a c t s using blood serum of two s e n s i t i v e i n d i v i d u a l s as the source of antibody. The immuno­ d i f f u s i o n r e a c t i o n between the peanut a l l e r g e n and a n t i b o d i e s i n t h e i r blood produced a sharp p r e c i p i t i n a r c , but the p r e c i p i t i n arc disappeared a f t e r s a l i n e d e p r o t e i n a t i o n of the gel on the g l a s s p l a t e . No p r e c i p i t i n arc occurred with blood from

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch018

286

X E N O B I O T I C S IN

FOODS A N D

FEEDS

nonsensitive individuals. We a l s o c o m p a r e d i m m u n o d i f f u s i o n o f i m m u n o g l o b u l i n s as a more p r e c i s e t e s t f o r e f f e c t s of p e a n u t a l l e r g e n s on i m m u n o g l o b u l i n s i n t h e i r b l o o d . T h i s method i s based on a r e p o r t by Kamat, et a l ( 1 1 ) , that b l o o d serum i m m u n o g l o b u l i n s from c o t t o n m i l l workers i n I n d i a and normal b l o o d s e r a showed s t r i k i n g d i f f e r e n c e s i n IgG l e v e l s . Normal s e r a h a d I g G l e v e l s o f 1400 mg. w h e r e a s s e r a of c o t t o n m i l l b y s s i n o t i c s a v e r a g e d 1850 mg. We e m p l o y e d t h i s t e c h n i q u e t o e x a m i n e b l o o d f r o m p e a n u t s e n s i t i v e i n d i v i d u a l s f o r IgA a n d IgG l e v e l s ( F i g u r e 1 ) . The gel c o n t a i n e d a n t i b o d i e s t o human I g A . B l o o d s e r a i n w e l l s were as follows: 1 , 5 , 9 , a r e s t a n d a r d IgA; 3 , 7 , 1 1 a r e serum of a n o n - s e n s i t i v e p e r s o n ; 2 , 6 , 1 0 a r e from s e n s i t i v e p a t i e n t P L ; and 4 , 8 , 1 2 a r e from s e n s i t i v e p a t i e n t CS. I m m u n o d i f f u s i o n was r u n o v e r n i g h t at 40C. T h e r e were s i g n i f i c a n t changes i n the s i z e of immunod i f f u s i o n r i n g s f o r b o t h s e n s i t i v e i n d i v i d u a l s compared to nonsensitive controls. Q u a n t i t y o f a s p e c i f i c i m m u n o g l o b u l i n was d e t e r m i n e d by m e a s u r i n g the d i a m e t e r of the r i n g and s u b s t i t u t i n g t h i s v a l u e i n a c h a r t w h i c h g i v e s t h e l e v e l of i m m u n o g l o b u l i n present i n that person's blood. In both p a t i e n t s examined, the I g A l e v e l s w e r e much l o w e r t h a n t h a t of t h e n o n s e n s i t i v e person and the IgA s t a n d a r d . I g G l e v e l s of t h e two p a t i e n t s w e r e s l i g h t l y h i g h e r t h a n t h e two n o n - a l l e r g i c c o n t r o l s . ( I n our t e s t s o n c o t t o n d u s t a n t i g e n s , I g G i n b l o o d of one b y s s i n o t i c p a t i e n t was c a l c u l a t e d t o b e 1 7 9 0 mg c o m p a r e d t o 1 2 9 2 mg f o r normal b l o o d , i n a c c o r d w i t h v a l u e s r e p o r t e d by Kamat, et a l (11).) Other a n t i n u t r i e n t s i n p e a n u t s , such as h e m a g g l u t i n i n s and t r y p s i n i n h i b i t o r s , a r e d e s t r o y e d by h e a t d u r i n g r o a s t i n g . Trypsin Inhibitors. T r y p s i n i n h i b i t o r a c t i v i t y i s found i n v i r t u a l l y a l l legumes, i n c l u d i n g soybeans and p e a n u t s . Trypsin i n h i b i t o r a c t i v i t y i n peanuts (PTI), l i k e that i n soybeans, can be d e t e c t e d by v a r i o u s m e t h o d s , most w h i c h m e a s u r e t h e a c t i v i t y of t r y p s i n on h y d r o l y s i s of a p r o t e i n or s y n t h e t i c s u b s t r a t e by spectrophotometer. We d e v e l o p e d a n i n d i r e c t i m m u n o c h e m i c a l i m m u n o e l e c t r o p h o r e t i c a n a l y s i s (IEA) method which d e t e c t s PTI i n m i c r o q u a n t i t i e s of peanut meals o r e x t r a c t s . I n s t e a d of m e a s u r i n g t h e i n h i b i t o r d i r e c t l y , as i s done f o r s o y b e a n t r y p s i n i n h i b i t o r , the i n d i r e c t method measures the e f f e c t of i n h i b i t o r f r a c t i o n s on t r y p s i n h y d r o l y s i s of a r a c h i n , the major peanut protein (Figure 2 J . The p r o t e i n s o u r c e , a r a c h i n , t h e m a j o r p e a n u t p e a n u t g l o b u l i n , was p l a c e d i n a l l w e l l s a n d a n t i b o d i e s to a r a c h i n was p l a c e d i n t h e t r o u g h s . I E A i n 1.5% i o n a g a r , p H 8 . 2 V e r o n a l b u f f e r , 0 . 2 5 M , was r u n f o r 2 h r a t 4 V / c m . For t r y p s i n + PTI i n t e r a c t i o n , m a t e r i a l s s t o o d 20 h r b e f o r e a d d i n g to a r a c h i n . For t r y p s i n + a r a c h i n , m a t e r i a l s stood 2 h r . After IEA, arcs were s t a i n e d w i t h Amido B l a c k . The i n d i r e c t method m e a s u r e s the e l e c t r o p h o r e t i c m i g r a t i o n of a r a c h i n u n t r e a t e d (upper slide), t r e a t e d w i t h t r y p s i n (second s l i d e ) and t r e a t e d w i t h t r y p s i n w h i c h was f i r s t i n c u b a t e d w i t h t h e p e a n u t i n h i b i t o r e x t r a c t

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch018

18.

ORY E TAL.

Antinutrients

and Allergens

in Oilseeds

287

Figure 1. Effect of peanut allergens on IgA levels in blood of sensitive individuals. Key: 1, 5, and 9, standard IgA; 3, 7, and 11, nonsensitive individual; 2, 6, and 10, sensitive patient PL; and 4, 8, and 12, sensitive patient CS.

Figure

2. Indirect

measurement of peanut electrophoretic

trypsin inhibitor analysis.

activity

by

immuno-

288

X E N O B I O T I C S IN F O O D S A N D

FEEDS

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch018

(third slide). A s shown h e r e , the i n h i b i t o r - t r e a t e d fraction shows a n i n c r e a s e i n m i g r a t i o n of a r a c h i n compared t o t h a t w i t h untreated trypsin. I f the t r y p s i n i n h i b i t o r i s heated before m i x i n g w i t h the t r y p s i n (the lower s l i d e ) , the i n h i b i t o r e f f e c t i s c o m p l e t e l y d e s t r o y e d , c o n f i r m i n g the heat s e n s i t i v i t y of PTI. Comparing PTI w i t h and w i t h o u t h e a t i n g and a d d i n g to t r y p s i n , i n d i c a t e s t h e s p e c i f i c i t y and p o t e n t i a l u s e f u l n e s s of t h i s method f o r s i m u l t a n e o u s a n a l y s i s of s e v e r a l peanut fractions. Hemagglutinins. Hemagglutinins are found i n v i r t u a l l y a l l o i l seeds and c e r e a l g r a i n s . D e c h a r y (6) isolated a hemagglutinin f r o m p e a n u t s t h a t was n o n s p e c i f i c f o r t y p e s A , B , a n d 0 r e d b l o o d cells. I t behaved i n a most u n u s u a l manner and i t s r a t h e r l o w p o t e n c y c o u l d n o t be i n c r e a s e d b y a n y o f t h e m e t h o d s available for fractionating peanuts. We c o n d u c t e d s i m i l a r s t u d i e s o n h e m a g g l u t i n i n s p u r i f i e d f r o m r i c e germ (RGA) by g e l electrophoresis, immunoelectrophoretic a n a l y s i s (IEA), red blood c e l l a g g l u t i n a t i o n t e s t s , a n d by c r o s s e d I E A , a more s e n s i t i v e t e s t t h a n one phase I E A , i f enough a n t i s e r u m i s a v a i l a b l e . Crossed IEA uses c o n s i d e r a b l y more a n t i s e r u m . F i g u r e 3 shows t h e e f f e c t s of the RGA o n human s e r u m p r o t e i n s . N o r m a l h u m a n s e r u m w i t h o u t RGA i s s h o w n i n t h e l e f t p l a t e a n d m i x e d w i t h RGA e x t r a c t f o r 1 h r . before crossed IEA i n f i r s t dimension i n the r i g h t p l a t e . Second dimension IEA (both plates) into a gel containing antibodies to human s e r u m p r o t e i n s was r u n f o r 2 h r . On t h e l e f t s i d e human s e r u m p r o t e i n s show t h e p r i m a r y s e r u m a l b u m i n ( t a l l peak i n f r o n t of the group) and the i m m u n o g l o b u l i n s (peaks c l o s e r to the origin). A f t e r e l e c t r o p h o r e s i s i n one d i r e c t i o n t h e g e l was electrophoresed i n the second d i r e c t i o n i n t o a g e l c o n t a i n i n g a n t i b o d i e s t o human s e r u m p r o t e i n s . Several proteins, including t h e f r o n t - r u n n i n g serum a l b u m i n and the s l o w e r i m m u n o g l o b u l i n s , now s h o w d e c r e a s e d m i g r a t i o n i n d i c a t i n g some t y p e o f b i n d i n g o f the a g g l u t i n i n to these p r o t e i n s . T h i s m e t h o d c a n be u s e d t o examine the e f f e c t of a g g l u t i n i n s on b i n d i n g to s p e c i f i c proteins in blood. Cottonseed (and Castor Seed). A l t h o u g h c o t t o n has been grown s i n c e a n c i e n t times for the f i b e r , the seed i s an i m p o r t a n t s o u r c e of v e g e t a b l e o i l and m e a l t o d a y . T h e m e a l , u s e d f o r many y e a r s a s a n i m a l f e e d , i s now b e i n g p r o p o s e d a s a new s o u r c e o f e d i b l e p r o t e i n f o r humans i f c e r t a i n h a r m f u l compounds p r e s e n t in s e e d s c a n be e l i m i n a t e d . Cottonseed meal c o n t a i n s storage p r o t e i n s and a l l e r g e n s . Cottonseed c o n t a i n s three major classes of p r o t e i n ; 2 S , 5 S , and 9S, i n a l m o s t e q u a l amounts. T h e 5S a n d 9S p r o t e i n s a r e s t o r a g e g l o b u l i n s w h e r e a s t h e 2 S p r o t e i n s are a l b u m i n s , sometimes c l a s s i f i e d as s t o r a g e p r o t e i n s because of t h e i r amino a c i d c o m p o s i t i o n , p r o p e r t i e s , and h i g h c o n c e n t r a t i o n s i n the seed (12).

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch018

18.

ORY E T AL.

Antinutrients

and Allergens

in

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The p r i m a r y a l l e r g e n s of c o t t o n s e e d , C S - 1 A , and c a s t o r s e e d , CB-1A, were p u r i f i e d s u f f i c i e n t l y from d e f a t t e d meals to p e r m i t d e t e r m i n a t i o n of t h e i r c h e m i c a l s t r u c t u r e ( 5 ) . They a r e s o l u b l e i n w a t e r a n d 25% e t h y l a l c o h o l b u t n o t i n 75% e t h y l a l c o h o l a n d are i m m u n o l o g i c a l l y d i s t i n c t from other a l l e r g e n s i n the seeds. They a r e not p r e c i p i t a t e d by b a s i c l e a d a c e t a t e , a r e s t a b l e i n b o i l i n g w a t e r , and r e s i s t d r a s t i c c h e m i c a l t r e a t m e n t . The C S - 1 A a l l e r g e n was l o c a l i z e d i n t h e p r o t e i n b o d i e s o f c o t t o n s e e d b y Y o u l e and Huang (12) u s i n g i m m u n o d i f f u s i o n of t h e a l l e r g e n and t h e 2 S , 5 S , a n d 9S p r o t e i n s o f t h e p r o t e i n b o d i e s a g a i n s t antib o d i e s to the CS-1A a l l e r g e n . They showed t h a t 2S a l b u m i n s c o n t a i n t h e c o t t o n s e e d a l l e r g e n , C S - 1 A , b y c r o s s r e a c t i n g t h e 2S albumins w i t h the a l l e r g e n . H o w e v e r , s i n c e t h e r e a r e no r e c o r d e d i n c i d e n t s of a l l e r g e n c i t y to c o t t o n s e e d p r o t e i n s by t h o s e c o n s u m i n g i t , the p r e s e n c e of t h i s a l l e r g e n i n the meal i s not a d e t e r r e n t to i t s a c c e p t a n c e as a source of p r o t e i n . A n o t h e r c o n c e r n today f o r a l l e r g e n s i n seeds i s the i n t e r e s t i n s p r o u t e d or p a r t l y germinated seeds ( e . g . : bean, a l f a l f a , or barley sprouts). T h e s e a r e n o t c o n s i d e r e d a l l e r g e n i c b u t we wondered a b o u t the e f f e c t of g e r m i n a t i o n on a l l e r g e n s . In g e r m i n a t i n g o i l s e e d s , the storage p r o t e i n s are m e t a b o l i z e d by p r o t e a s e s , but the a l l e r g e n s are not. We e x a m i n e d t h e e f f e c t s of seed g e r m i n a t i o n on the a l l e r g e n CB-1A i n c a s t o r seeds (the c o u n t e r p a r t of CS-1A i n c o t t o n s e e d ) . Many a l l e r g e n s a r e not d i g e s t e d by p r o t e a s e s i n t h e a l i m e n t a r y s y s t e m , w h i c h p r e v e n t s t h e i r m e t a b o l i s m by n o n - r u m i n a n t s . The CB-1A a l l e r g e n m a i n t a i n e d i t s a n t i g e n i c s t r u c t u r e up t o t e n d a y s o f g e r m i n a t i o n t o c o n f i r m i n v i t r o t e s t s on the e f f e c t of p r o t e o l y t i c enzymes on a l l e r g e n s , w h i c h showed t h e y a r e not m e t a b o l i z e d by p r o t e a s e s (13). W h i l e c a s t o r seeds a r e i n d u s t r i a l l y i m p o r t a n t as a source of o i l , the o i l or p r o t e i n i s not e d i b l e because s e v e r a l potent a l l e r g e n s are present i n the seed. The seed c o n t a i n s a p p r o x i m a t e l y 5 0 % o i l a n d 18% p r o t e i n b u t a l s o c o n t a i n s t h e t o x a l b u m i n r i c i n , the CB-1A s e r i e s of a l l e r g e n s , and a n a l k a l o i d , ricinine. R i c i n , molecular weight 40,000, i s an extremely potent p h y t o t o x i n , the most p o t e n t x e n o b i o t i c y e t d i s c o v e r e d i n oilseeds. E v e n c r u s h i n g of c a s t o r seeds to s e p a r a t e the o i l f o r i n d u s t r i a l u s e s c a n be q u i t e h a z a r d o u s . Ricin is water-soluble and i s present i n the dust generated d u r i n g c r u s h i n g . Constant i n h a l a t i o n of the dust can cause s e r i o u s problems u n l e s s a i r - f i l t e r i n g masks and o t h e r p r e c a u t i o n s a r e t a k e n . Ricin, like the c a s t o r a l l e r g e n , i s not d i g e s t e d by mammalian p r o t e o l y t i c enzymes. Gossypol. Gossypol i s another substance i n cottonseed that p r e v e n t s g r e a t e r u t i l i z a t i o n of the m e a l by n o n - r u m i n a n t s . It is a p o l y p h e n o l i c g r e e n i s h - y e l l o w pigment present w i t h i n glands i n g l a n d e d c o t t o n s e e d , t h a t c a n r e d u c e the n u t r i t i v e v a l u e of t h e meal. The s t r u c t u r e of g o s s y p o l i s shown i n F i g u r e 4 . Gossypol i s not p r e s e n t i n the newer v a r i e t y of g l a n d l e s s s e e d . The

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Figure

3. Crossed

immunoelectrophoretic analysis agglutinin and human serum

Figure

4. Chemical

structure

of

of binding proteins.

gossypol.

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pigment g l a n d s appear as d a r k p u r p l i s h - b l a c k d o t s t h r o u g h o u t t h e endosperm. G o s s y p o l g l a n d s v a r y i n s i z e f r o m 1 0 0 t o kOO μ . From 10 t o 40 g l a n d s a r e f o u n d i n a c r o s s s e c t i o n or a s e e d k e r n e l . W h e n t h e s e e d s a r e c r u s h e d t o p r o d u c e t h e o i l , g o s s y p o l c a n be l e a c h e d i n t o t h e o i l o r c a n be r e t a i n e d b y t h e m e a l . If retained by the m e a l , the meal i s u n s u i t a b l e as a s o u r c e of f e e d f o r n o n ruminants. G o s s y p o l o c c u r s i n b o t h f r e e and bound forms. Interr e a c t i o n of g o s s y p o l w i t h p r o t e i n s t a k e s p l a c e t h r o u g h the a l d e ­ hyde g r o u p s of g o s s y p o l and the amino g r o u p s of f r e e amino a c i d s or t e r m i n a l amino groups of p o l y p e p t i d e s i n the p r o t e i n . The most f r e q u e n t l y bound amino a c i d i s l y s i n e , w h i c h l o w e r s nutri­ t i v e v a l u e of the p r o t e i n . The e a r l i e s t e f f e c t s of g o s s y p o l were f o u n d on h a t c h a b i l i t y o f e g g s and on egg d i s c o l o r a t i o n ( 1 4 ) . C h i c k s fed cottonseed meal c o n t a i n i n g h i g h g o s s y p o l p r o d u c e d s m a l l e r eggs and eggs w i t h decreased h a t c h a b i l i t y . Egg l a y e r s p r o d u c e d eggs w h i c h had d i s ­ colored yolks. T h i s e f f e c t ( t h e H a l p h e n r e a c t i o n ) was believed t o be due t o r e a c t i o n o f g o s s y p o l w i t h i r o n i n t h e egg y o l k t h a t p r o d u c e d a g r e e n i s h y o l k i n s t e a d of y e l l o w . R e a c t i o n of gossypol w i t h i r o n c a n p r o d u c e low serum l e v e l s of i r o n and l o w u p t a k e of i r o n by m o n o g a s t r i c s . Gossypol can a l s o i n h i b i t various enzymes. T a n k s l e y et a l . (15) found that g o s s y p o l prevented the c o n v e r s i o n of p e p s i n o g e n t o p e p s i n . They i n c u b a t e d p e p s i n o g e n w i t h 2 : 1 o r 3 : 1 m o l a r r a t i o s of g o s s y p o l and showed t h a t t h e c o n v e r s i o n of p e p s i n o g e n t o p e p s i n was b l o c k e d a t l o w p H v a l u e s . T h i s was t h e f i r s t r e p o r t of a n a t u r a l l y o c c u r r i n g i n h i b i t o r f o r the conver­ s i o n of a n i n a c t i v e zymogen to i t s a c t i v e f o r m . Gossypol also i n h i b i t s a c t i v i t y o f A T P a s e a n d s u c c i n a t e d e h y d r o g e n a s e JLn v i t r o (16). The e f f e c t of g o s s y p o l - a c e t i c a c i d on plasma l i p i d c o n c e n ­ t r a t i o n was r e c e n t l y s t u d i e d i n a d u l t m o n k e y s ( 1 7 ) . Significant decreases i n t o t a l plasma c h o l e s t e r o l , low d e n s i t y lipoprotein ( L D L ) , and v e r y low d e n s i t y l i p o p r o t e i n - c h o l e s t e r o l were observed w i t h o u t any s i g n i f i c a n t d e c r e a s e i n plasma h i g h d e n s i t y lipopro­ t e i n c h l o r e s t e r o l l e v e l s i n monkeys g i v e n 10 mg/kg/day. This t h e r a p e u t i c p r o p e r t y of g o s s y p o l has not been p r e v i o u s l y report­ ed. I t i s tempting to s p e c u l a t e that gossypol might p o s s i b l y reduce i n t e s t i n a l a b s o r p t i o n of d i e t a r y c h o l e s t e r o l o r h e p a t i c s y n t h e s i s of LDL i n mammals. I n a d d i t i o n t o i t s a d v e r s e e f f e c t s on mammals, g o s s y p o l has a l s o b e e n shown t o be t o x i c t o i n s e c t s . Among t h e pesticides shown t o m a n i f e s t p l a n t r e s i s t a n c e a r e c e r t a i n c l a s s e s of pheno­ l i c compounds, i n c l u d i n g g o s s y p o l , f l a v o n o i d s , and a r o m a t i c a c i d s (18). The r o l e s of t h e s e compounds a r e d i f f i c u l t t o e l u c i d a t e b e c a u s e c o m p o u n d s t o x i c t o o n e i n s e c t may n o t b e t o x i c to a n o t h e r , b u t g l a n d l e s s c o t t o n s e e d i s s i g n i f i c a n t l y more s u s c e p ­ t i b l e to i n s e c t a t t a c k than glanded c o t t o n s e e d . Gossypol i n c o t t o n a c t s as an i n h i b i t o r of i n s e c t s b u t the r e s i s t a n c e has not been c o r r e l a t e d w i t h the t o t a l g o s s y p o l . E a g l e (19) concluded

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t h a t f r e e g o s s y p o l content of c o t t o n s e e d meal i s not a t r u e m e a s u r e o f m e a l t o x i c i t y , a l t h o u g h many o t h e r s c o n s i d e r t o t a l gossypol as the primary t o x i c agent. P e r h a p s t h e most i n t e r e s t i n g e f f e c t of g o s s y p o l today i s i t s use i n C h i n a as a male c o n t r a c e p t i v e . G o s s y p o l was f i r s t noted t o a f f e c t f e r t i l i t y a s l o n g as twenty y e a r s ago b u t c l i n i c a l t r i a l s o n l y began about 1972 ( 2 0 ) . Up t o 1 9 7 2 , more t h a n 1 0 , 0 0 0 men h a d b e e n s t u d i e d o v e r l o n g p e r i o d s . Each received d a i l y o r a l d o s e s o f 2 0 mg u n t i l s p e r m c o u n t w a s s u f f i c i e n t l y r e d u c e d (about t w o m o n t h s ) , w i t h m a i n t e n a n c e d o s e s o f 7 5 - 1 0 0 mg t a k e n t w i c e monthly. Side e f f e c t s were m i n i m a l and sperm c o u n t s r e t u r n e d t o n o r m a l w i t h i n a few months a f t e r u s e o f g o s s y p o l was d i s c o n t i n u ed. T h e m e c h a n i s m o f a c t i o n was f o u n d t o be i n h i b i t i o n o f l a c tate dehydrogenase-X, a n enzyme f o u n d o n l y i n s p e r m a n d t e s t i s cells. Cyclopropene Fatty Acids C y c l o p r o p e n e f a t t y a c i d s (CPFA) a r e p r e s e n t i n c o t t o n s e e d o i l . They c a n a f f e c t l i p i d m e t a b o l i z i n g enzymes i n a n i m a l s by c h a n g i n g t h e o l e i c : l i n o l e i c f a t t y a c i d r a t i o towards higher s a t u r a t i o n (21). The a b s o l u t e amounts of o l e i c a n d l i n o l e i c a c i d s d e c r e a s e a n d more s t e a r i c (saturated C-18 f a t t y a c i d ) i s produced. W i t h a l i p a s e prepared from c a s t o r s e e d s , we s h o w e d t h a t C P F A i n h i b i t e d a c t i v i t y b y f o r m i n g a c o v a l e n t bond between t h e SH-group of c y s t e i n e a t the a c t i v e site o f t h e enzyme ( 2 2 ) . In v i t r o tests with the SH-sensitive lipase of c a s t o r seeds showed t h a t c o t t o n s e e d o i l c o n t a i n i n g m a l v a l i c a c i d , a C P F A , r e d u c e d l i p a s e a c t i v i t y u n l e s s c y s t e i n e was a d d e d first. F r e e c y s t e i n e bound t o CPFA p r e f e r e n t i a l l y , reducing i n h i b i t i o n of t h e enzyme ( 2 2 ) . I n c o n c l u s i o n , many o i l s e e d s a n d c e r e a l g r a i n s b e i n g c o n s i d e r ed a s s o u r c e s o f p r o t e i n f o r f u t u r e f o o d p r o d u c t s c o n t a i n c o m pounds w i t h a l l e r g e n i c o r a n t i n u t r i e n t p r o p e r t i e s . Unless precaut i o n s a r e t a k e n t o remove o r d e t o x i f y such compounds, u n d e s i r a b l e effects can occur. However, w i t h the technology a v a i l a b l e f o r food p r o c e s s i n g today, our food supply i s q u i t e safe and s h o u l d not present s e r i o u s problems f o r consumers.

Literature Cited 1. 2. 3. 4. 5. 6. 7.

Anon. Food & Nutrition News 1979, 50 (2), 4. von Pirquet, C. Muenchen. Med. Woechenschr. 1906, 53, 1457. Speer, F. Amer. Family Physician 1976, 13, 106. Aas, K. Clinical Allergy 1978, 8, 39. Spies, J.R.; Coulson, E . J . ; Chambers, D.C.; Bernton, H.S.; Stevens, H.; Shimp, J.H. J . Amer. Chem. Soc. 1951, 73, 3995. Dechary, J.M.; Leonard, G.L.; Corkern, S. Lloydia 1970, 33, 270. Frampton, V.L.; Boudreaux, H.B. Econ. Bot. 1963, 17, 312.

18.

8. 9. 10. 11. 12. 13.

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14. 15. 16. 17. 18. 19. 20. 21. 22.

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Ory, R.L.; Neucere, N.J. J. Amer. Peanut Res. and Education Assoc. 1971, 3, 57. Gillespie, D.N.; Nakajima, S.; Gleich, G.J. J. Allergy Clin. Immunol. 1976, 57, 302. Sachs, M.I,; Jones, R.T.; Yunginger, J.N.; Gleich, G.J. J. Allergy Clin. Immunol. 1979, 63, 197. Kamat, S.R.; Tasker, S.P.; Iyer, E.R.; Naik, M. J. Soc. Occup. Med. 1979, 29, 102. Youle, R.J.; Huang, A.H.C. J. Agric. Food Chem. 1979, 27, 500. Daussant, J.; Ory, R.L.; Layton, L.L. J. Agric. Food Chem. 1976, 2A, 103. Adams, R.; Geissman, T.A.; Edwards, J.D. Chem. Rev. 1960, 60, 555. Tanksley, T.D.; Neumann, H.; Lyman, C.M.; Pace, C.N.; Prescott, J.M. J. Biol. Chem. 1970, 245, 6456. Kalla, N.R.; Tet Wei, J.F. IRCS Med. Sci. Libr. Compend. 1981, 9, 792. Shandilya, L.N.; Clarkson, T.B. Lipids 1982, 17, 285. Hedin, P.A. J. Agric. Food Chem. 1982, 30, 201. Eagle, E.; Davies, D.L. J. Amer. Oil Chem. Soc. 1958, 35, 36. Maugh, T.H. Science 1981, 212, 314. Reiser, R., Raju, P. K. Biochem. Biophys. Res. Commun. 1964, 17, 8. Ory, R.L.; Altschul, A.M. Biochem. Biophys. Res. Commun. 1964, 17, 12.

RECEIVED May 13, 1983

19 Psoralens as Phytoalexins in Food Plants of the Family Umbelliferae Significance in Relation to Storage and Processing

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch019

ROSS C. BEIER, G. WAYNE IVIE, and ERNEST H. OERTLI Veterinary Toxicology and Entomology Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture, College Station, TX 77841 Linear furanocoumarins (psoralens) are phototoxic, photomutagenic, and photocarcinogenic compounds that occur as natural constituents of hundreds of plant species, including some food plants of the family Umbelliferae (e.g., parsnip, celery, and parsley). Certain plant stresses, particularly diseases, induce biosynthesis of toxic natural plant products; a phenomenon referred to as a phytoalexin response. Such interactions in food plants of the family Umbelliferae may have toxicological implications for man because of the biological activity of psoralens. The linear furanocoumarin phytoalexin response in celery is discussed, with brief comments concerning carrots and parsley. Historical Psoralens. Many plants contain linear furanocoumarins (psoralens) (J_), which were i d e n t i f i e d i n the l a t e 1940 s as the cause of the p h o t o s e n s i t i z a t i o n properties of these plants (_2"_4) · Plants containing l i n e a r furanocoumarins can cause p h o t o s e n s i t i z a t i o n i n l i v e s t o c k and poultry (_5-9_), r e s u l t i n g i n economic losses and, i n some cases, animal death. Man has also encountered problems with the p h o t o s e n s i t i z i n g properties of l i n e a r furanocoumarins. Celery handlers and f i e l d workers are frequently affected with p h o t o s e n s i t i z a t i o n of the f i n g e r s , hands, and forearms (10,11). These s k i n disorders are r e f e r r e d to as c e l e r y d e r m a t i t i s , c e l e r y i t c h , or c e l e r y b l i s t e r s , and are caused by l i n e a r furanocoumarins i n diseased c e l e r y (12). Some researchers (12,13) have found l i n e a r furanocoumarins only i n diseased p l a n t s , whereas others (14,15) obtained them from healthy celery. f

T h i s chapter not subject t o U . S . copyright. P u b l i s h e d 1983, A m e r i c a n C h e m i c a l S o c i e t y

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Biological Activities of Linear Furanocoumarins» The b i o l o g i c a l a c t i v i t y of these compounds are extremely d i v e r s e because of t h e i r i n t e r a c t i o n s with DNA (32-35), and RNA (36). L i n e a r furanocoumarins have antifeedant a c t i v i t y toward various insects (37,38) and are phototoxic to others (39). I n t e r e s t i n g l y , the black s w a l l o w t a i l b u t t e r f l y has maximized i t s metabolic d e t o x i f i c a t i o n processes allowing i t s l a r v a to feed on plants with a high l i n e a r furanocoumarin content (40). Psoralen, bergapten, xanthotoxin, and isopimpinellin are a n t i b a c t e r i a l when combined with UV l i g h t , whereas psoralen and xanthotoxin have some a n t i b a c t e r i a l a c t i v i t y without UV l i g h t (41). A mixture of p i m p i n e l l i n , i s o p i m p i n e l l i n , isobergapten, and sphondin was fungi t o x i c at 200 ppm or less (42). The individual linear furanocoumarins, psoralen (43), and xanthotoxin (44,45) are also a n t i f u n g a l . Toxicological Implications f o r Man. Because psoralens are potent photoactive compounds, they have been used m e d i c a l l y f o r treatment of skin depigmentation or vitiligo (16,17), and psoriasis (18). However, there has r e c e n t l y been concern a s s o c i a t e d with the medical use of these compounds (19) · This concern i s due to the observed p h o t o t o x i c i t y during therapeutic use (17), the suspected p h o t o c a r c i n o g e n i c i t y of xanthotoxin (20,21), and the l a t e n t onset of tumors i n treated laboratory animals (22). Acute gout secondary to p s o r i a s i s also was exacerbated by psoralen and UV-A (PUVA) photochemotherapy (23). Psoralens i n Healthy Celery. Healthy c e l e r y contains at l e a s t four l i n e a r furanocoumarins (Figure 1), psoralen, bergapten, xanthotoxin, and i s o p i m p i n e l l i n (14). The observed q u a n t i t i e s of l i n e a r furanocoumarins i n healthy samples of three d i f f e r e n t c e l e r y c u l t i v a r s grown at d i f f e r e n t l o c a t i o n s i n the U.S. are shown i n Table I . Table I . Summary of the L i n e a r Furanocoumarin Content i n Fresh Celery Grown i n C a l i f o r n i a , F l o r i d a , and M i c h i g a n 3

b , c

Compound

A

Cultivar Β

Psoralen Bergapten Xanthotoxin Isopimpinellin

0.15 + 0.06 0.14 + 0.04 0.61 + 0.14 0.08+0.03

. s c l e r o t i o r u m i s often implicated i n the production of linear furanocoumarins i n celery, s t e r i l i z i n g the plant tissues maybe an appropriate first step in many investigations.

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26°C

il 2°C

Φ

Q

10

Time

15

"Ί 20

(min)

Figure 5. HPLC tracings of detector response vs. retention time for UV-treated celery cv. 5270-R after 72 h at 2 °C and 26 °C in comparison to the linear fur­ anocoumarin standards: psoralen (p), bergapten (b), xanthotoxin (x), and isopim­ pinellin (i).

19.

BEIER E T A L .

Phytoalexins

in Food

305

Plants

Celery p e t i o l e s were immersed i n 0.1% sodium h y p o c h l o r i t e f o r 20 min. and incubated f o r 72 hrs at 26° C. Subsequent e x t r a c t i o n and HPLC a n a l y s i s gave the t r a c i n g s shown i n Figure 6. The p s o r a l e n l e v e l s at 2°C are s i m i l a r to the CuSO^ t r e a t e d c e l e r y , but at 26° C the l e v e l s are lower and appear qualitatively different from those observed in the CuSO^-treated plant m a t e r i a l . A composite bar graph showing the l e v e l s of the four linear furanocoumarins in both UV-treated and sodium h y p o c h l o r i t e treated c e l e r y analyzed a f t e r 72 hrs at 26°C i s shown i n Figure 7. The observed l e v e l s i n both treated t i s s u e s were s i g n i f i c a n t l y higher than those i n the c o n t r o l s .

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch019

Summary The total quantities of psoralens and t h e i r increased concentration i n c e l e r y a f t e r d i f f e r e n t treatments i s described i n Table 3.

Table 3.

Summary of P h y t o a l e x i n Response i n Treated C e l e r y (57)

Treatment

Time ( h r )

T o t a l Psoralens (ppm)

F o l d Increase

104.0 23.0 3.4 2.2 8.8

CuSO^ CuSO^ UV l i g h t Sodium h y p o c h l o r i t e Cold CuS0 *

96 79 72 72 72 48

26.0 21.1 7.4 4.9 2.2 29.0

4-day-old CuSO/,*

48

1.3

4

-23.0

*These experiments were c a r r i e d out on c e l e r y procured a t the same time. Part of the l o t was immediately t r e a t e d , while another p o r t i o n was r e t a i n e d f o r 4 days i n the r e f r i g e r a t o r before treatment.

A l l of the treatments i n Table 3 caused an increase i n the q u a n t i t i e s of l i n e a r furanocoumarins to some degree, with some samples containing as much as 29 ppm of t o t a l psoralens. I t i s a l s o i n t e r e s t i n g that a sample of harvested c e l e r y allowed to age 4 days i n the l a b o r a t o r y prior to CuSO^ treatment e x h i b i t e d a 23 f o l d decrease i n the t o t a l l i n e a r furanocoumarin production when compared to non-aged CuSO^-treated celery (Table 3 ) . This r e s u l t may r e f l e c t a d e t e r i o r a t i o n of the c e l l u l a r c o n d i t i o n i n these samples.

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch019

306

XENOBIOTICS

I

1—ι

0

5

10

IN FOODS A N D F E E D S

1—I 15

20

Time (min) Figure 6. HPLC tracings of the detector response vs. retention time for sodium hypochlorite-treated celery cv. 5270-R after 72 h at 2 °C and 26 °C in comparison to the linear furanocoumarin standards: psoralen (p), bergapten (b), xanthotoxin (x), and isopimpinellin (i).

Psoralens from celery cv. 5270-R Figure 7. A bar graph of the observed levels of psoralen, bergapten, xanthotoxin, and isopimpinellin in celery cv. 5270-R 72 h after UV light and sodium hypo­ chlorite treatment.

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch019

19.

BEiER E T A L .

Phytoalexins

in Food

Plants

307

What i s the Impact of the Phytoalexin Response i n Celery. Studies to date i n d i c a t e that c e l e r y purchased at l o c a l markets should contain low l e v e l s of linear furanocoumarins (14). Increased l e v e l s of psoralens as a r e s u l t of a phytoalexin response w i l l probably be of l i t t l e or no s i g n i f i c a n c e to the consumer. The major i n t e r e s t that a r i s e s from t h i s phenomenon will be to the grower, f i e l d worker and celery handler. C l e a r l y , c e r t a i n chemicals and s t r e s s s i t u a t i o n s can cause up to 100 f o l d increases i n the l i n e a r furanocoumarin content of previously excised celery petioles. Therefore, i t seems possible that certain stress situations and/or chemical treatments may indeed elevate l e v e l s of psoralens i n c e l e r y to a point where the r i s k of d e r m a t i t i s i s g r e a t l y enhanced. The a c t u a l r o l e of l i n e a r furanocoumarins i n the disease r e s i s t a n c e of c e l e r y i s unknown; however, i t has been concluded that the phytoalexins studied to date play an important r o l e i n r e s i s t a n c e (47). Phytoalexins

i n Other

umbelliferae?

Carrots. Previous phytochemical studies with carrot (Daucus carota L.) have been unsuccessful i n demonstrating the presence of psoralens, and i t i s g e n e r a l l y accepted that c a r r o t s lack l i n e a r furanocoumarins (58,59). We have r e c e n t l y developed techniques to look f o r psoralens at the sub p a r t s - p e r - m i l l i o n l e v e l i n carrot (60), and were s i m i l a r l y unable to see l i n e a r furanocoumarins i n t h i s p l a n t . Along with our c e l e r y s t u d i e s , we treated c a r r o t s l i c e s with CuS0 (9 Χ 10"" M) f o r 0.5 hr, and made analyses by HPLC a f t e r 72 h r s . Even with t h i s attempted s t i m u l a t i o n , no l i n e a r furanocoumarins were detected. I t has been suggested (46) that phytoalexins can be used i n some cases as taxonomic markers. That a p p l i c a t i o n may indeed be appropriate i n the case of c a r r o t s . 3

4

Parsley. P a r s l e y (Petroselinum sativum) has been known to cause d e r m a t i t i s on the hands and arras. This c o n d i t i o n was accompanied by b l i s t e r s which developed on the back of the hands of s c h o o l g i r l s that picked parsley. Peasants i n a v i l l a g e near S o f i a , B u l g a r i a , are f a m i l i a r with t h i s problem, and some cover t h e i r hands with f a t before p i c k i n g (61). The first linear furanocoumarin to be isolated from p a r s l e y was bergapten (62). Later work provided quantitative data f o r bergapten, xanthotoxin, and i s o p i m p i n e l l i n from d r i e d p a r s l e y grown i n greenhouse conditions (15). We are presently investigating parsley for i t s linear furanocoumarins besides those previously identified. Preliminary studies with CuSO^ suggests that parsley also produces psoralens as phytoalexins.

308

XENOBIOTICS IN FOODS A N D F E E D S

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch019

Literature Cited 1. Nielsen, Β. E. "The Biology and Chemistry of the Umbelliferae"; Heywood, V. H., Ed.; Academic Press: London, 1971, p. 325. 2. Fahmy, I. R.; Abushady, H.; Schonberg, Α.; Sina, A. Nature 1947, 160, 468-9. 3. Fahmy, I. R.; Abu-Shady, H. Quart. J . Pharm. Pharmacol. 1947, 20, 281-91. 4. Fahmy, I. R.; Abu-Shady, H. Q. J . Pharm. Parmacol. 1948, 21, 499-503. 5. Binns, W.; James, L. F.; Brooksby, W. Vet. Med. Small Anim. Clin. 1964, 59, 375-9. 6. Musajo, L.; Rodighiero, G. Phytophysiology 1972, 7, 115-47. 7. Stegmaier, O. C. J . Invest. Dermatol. 1959, 32, 345-9. 8. Dollahite, J . W.; Younger, R. L.; Hoffman, G. O. Am. J . Vet. Res. 1978, 39, 193-7. 9. Egyed, M. N.; Shlosberg, Α.; Eilat, Α.; Cohen, U.; Beemer, A. Refu. Vet. 1974, 31, 128-31. 10. Birmingham, D. J . ; Key, M. M.; Tubich, G. E.; Perone, V. B. Archs. Derm. 1961, 83, 73-85. 11. Legrain, P. MM.; Barthe, R. Bull. Soc. Fr. Derm. Syph. 1926, 33, 662-4. 12. Scheel, L. D.; Perone, V. B.; Larkin, R. L.; Kupel, R. E. Biochemistry 1963, 2, 1127-31. 13. Wu, C. M.; Koehler, P. E.; Ayres, J . C. Appl. Microbiol. 1972, 23, 852-6. 14. Beier, R. C.; Ivie, G. W.; Oertli, Ε. H.; Holt, D. L. Food Chem. Toxicol. 1983, 21, 163-5. 15. Innocenti, G.; Dall'Acqua, F.; Caporale, G. Planta Medica 1976, 29, 165-70. 16. Scott, B. R.; Pathak, Μ. Α.; Mohn, G. R. Mutat. Res. 1976, 39, 29-74. 17. Pathak, M. Α.; Daniels, F.; Fitzpatrick, T. B. J . Invest. Dermatol. 1962, 39, 225-49. 18. Van Scott, E. J. Am. Med. Assoc. 1976, 235, 197-8. 19. "Psoralens," National Toxicology Program Technical Bulletin, 1982, 6, p. 8. 20. Reed, W. B. Acta Derm.-Venereol. 1976, 56, 315-7. 21. Stern, R. S.; Thibodeau, L. Α.; Kleinerman, R. Α.; Parrish, J . A.; Fitzpatrick, T. B.; 22 participating investigators. N. Eng. J . Med. 1979, 300, 809-13. 22. Zajdela, F.; Bisagni, E. Carcinogenesis 1981, 2, 121-7. 23. Burnett, J . W. Arch. Dermatol. 1982, 118, 211. 24. Johnson, C.; Brannon, D. R.; Kuć, J . Phytochemistry 1973, 12, 2961-2. 25. Müller, K.; Börger, H. Arb. Biol. Reichsanstat. Land-u Forstwirtsch. 1940, 23, 189-231. 26. Müller, Κ. Phytopathol. Ζ. 1956, 27, 237-54.

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch019

19.

BEIER E T A L .

Phytoalexins

in Food

Plants

309

27. Kuć, J . Annu. Rev. Phytopathol. 1972, 10, 207-32. 28. Paxton, J . D. Pl. Disease 1980, 64, 734. 29. Smith, D. A. "Phytoalexins"; Bailey, J. Α.; Mansfield, J. W., Eds.; John Wiley and Sons: New York, 1982, p. 218. 30. Henry, S. Α.; Cantab, M. D.; Cantab, D.P.H. Br. J . Derm. 1933, 45, 301-9. 31. Marasas, W.F.O.; van Rensburg, S. J. "Plant Disease"; Horsfall, J. G.; Cowling, Ε. Β., Eds.; Academic Press: New York, 1979, Vol. IV, p. 368. 32. Parsons, B. J . Photochemistry and Photobiology 1980, 32, 813-21. 33. Grekin, D. Α.; Epstein, J. H. Photochemistry and Photobiology 1981, 33, 957-60. 34. Belogurov, Α. Α.; Zavilgelsky, G. B. Mutation Research 1981, 84, 11-5. 35. Cassier, C.; Moustacchi, E. Mutation Research 1981, 84, 37-47. 36. Talib, S.; Banerjee, A. K. Virology 1982, 118, 430-8. 37. Tajima, T.; Munakata, K. Agric. Biol. Chem. 1979, 43, 1701-6. 38. Muckensturm, B.; Duplay, D.; Robert, P. C.; Simonis, M. T.; Kienlen, J . C. Biochemical Systematics and Ecology 1981, 9, 289-92. 39. Berenbaum, M. Ecology 1981, 62, 1254-66. 40. Ivie, G. W.; Bull, D. L.; Beier, R. C.; Pryor, N. W.; Oertli, Ε. H. Science 1983 (In press). 41. Fowles, W. L.; Griffith, D. G.; Oginsky, E. L. Nature 1958, 181, 571-2. 42. Martin, J . T.; Baker, Ε. Α.; Byrde, R.J.W. Ann. Appl. Biol. 1966, 57, 501-8. 43. Stanley, W. L.; Jund, L. J. Agric. Food Chem. 1971, 19, 1106-10. 44. Knudsen, E. A. Acta Derm. (Stockholm) 1980, 60, 452-6. 45. Oberste-Lehn, H.; Plempel, M. Dermatologica 1977, 154, 193-202. 46. Grisebach, H.; Ebel, J . Angew. Chem. Int. Ed. Engl. 1978, 17, 635-47. 47. Bell, A. A. Annu. Rev. Plant Physiol. 1981, 32, 21-81. 48. Hargreaves, J . A. Physiol. Plant Path. 1979, 15, 279-87. 49. Carlson, R. E.; Dolphin, D. H. Phytοchemistry 1981, 20, 2281-4. 50. Reilly, J . J . ; Klarman, W. L. Phytopathology 1972, 62, 1113-5. 51. Oku, H.; Nakanishi, T.; Shiraishi, T.; Ouchi, S. Sci. Rep. Fac. Agric., Okayama Univ. 1973, 42, 17-20. 52. Hadwiger, L. Α.; Jafri, Α.; von Broembsen, S.; Eddy, R., Jr. Plant Physiol. 1974, 53, 52-63. 53. Rahe, J . E.; Arnold, R. M. Can. J. Bot. 1975, 53, 921-8. 54. Hadwiger, L. Α.; Schwochau, M. E. Can. J . Bot. 1971, 47, 588-90.

310

55. 56. 57. 58. 59. 60. 61.

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch019

62.

XENOBIOTICS IN F O O D S A N D F E E D S

Bridge, Μ. Α.; Klarman, W. L. Phytopathology 1972, 63, 606-9. Ivie, G. W. J. Agric. Food Chem. 1978, 26, 1394-1403. Beier, R. C.; Oertli, Ε. H. Phytochemistry 1983 (In press). Berenbaum, M.; Feeny, P. Science 1981, 212, 927-9. Ivie, G.; Holt, D.; Ivey, M. Science 1981, 213, 909-10. Ivie, G. W.; Beier, R. C.; Holt, D. L. J . Agric. Food Chem. 1982, 30, 413-6. Stransky, L.; Tsankov, N. Contact Dermatitis 1980, 6, 233-4. Musajo, L.; Caporale, G.; Rodighiero, G. G. Gazz. Chim. Ital. 1954, 84, 870-3.

RECEIVED June 28,

1983

20 Food, Drug, and Cosmetic Colors: Toxicological Considerations JOSEPH F. BORZELLECA Department of Pharmacology and Toxicology, Medical College of Virginia, Virginia Commonwealth University, Richmond, VA 23298

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch020

JOHN HALLAGAN and CAROLINE REESE The Offices of Daniel R. Thompson, 900 17th St. N.W., Washington, DC 20006

The Food, Drug and Cosmetic (FD&C) Colors have been studied extensively. The most recent have been chronic toxicity/oncogenicity studies in Charles River CD rats and Charles River CD-1 mice. The colors evaluated and the dietary levels employed were: Dietary Levels (%) Color Rat Mouse FD&C Blue 1 0.0, 0.1, 1.0, 2.0 0.0, 0.5, 1.5, 5.0 FD&C Green 3 0.0, 1.25, 2.5, 5.0 0.0, 0.5, 1.5, 5.0 FD&C Blue 2 0.0, 0.5, 1.0, 2.0 0.0, 0.5, 1.5, 5.0 FD&C Yellow 6 0.0, 0.75, 1.5, 3.0, 5.0 0.0, 0.5, 1.5, 5.0 FD&C Yellow 5 0.0, 0.1, 1.0, 2.0, 5.0 0.0, 0.5, 1.5, 5.0 FD&C Red 3 0.0, 0.1, 0.5, 1.0, 4.0 0.0, 0.3, 1.0, 3.0 Many parameters were measured during the course of these studies including reproductive performance (rats only), body weights, feed consumption, clinical chemistries (blood, urine), hematology, gross and histological examination of tissues. Data were subjected to extensive statistical analyses. There were no consistent statistically significant and biologically relevant compound related effects, including tumors. These studies were supported by the Certified Colors Manufacturers Association and were conducted by Bio-dynamics Laboratory and International Reseach and Development Corporation. C o l o r s have been an e s s e n t i a l p a r t o f o u r e x i s t e n c e . N a t u r e i s c o l o r f u l . C o l o r s have been used t h r o u g h o u t h i s t o r y b y man i n a t l e a s t t h r e e m a j o r a r e a s - f o o d , d r u g s and c o s m e t i c s . C o l o r e d c a n d y h a s been i d e n t i f i e d i n p a i n t i n g s i n E g y p t i a n tombs d a t i n g 0097-6156/83/0234-0311$06.50/0 © 1983 A m e r i c a n C h e m i c a l S o c i e t y

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch020

312

X E N O B I O T I C S IN F O O D S A N D

FEEDS

back t o a b o u t 1500 B.C. A r t i f i c i a l l y c o l o r e d w i n e s were r e p o r t e d b y P l i n y t h e E l d e r i n a b o u t 400 B.C. Ebers Papyrus, t h e o l d e s t r e c o r d o f d r u g s , i d e n t i f i e s c o l o r s t h a t were used i n drugs. A b o u t 5000 B.C., E g y p t i a n s were u s i n g g r e e n c o p p e r f o r eye shadow. A l s o u s e d were henna t o dye h a i r , c a r m i n e t o r e d d e n l i p s , k o h l (an a n t i m o n y compound) t o b l a c k e n e y e b r o w s , l i d s a n d l a s h e s . A r o u n d 500 B.C., s a f f r o n was u s e d t o make f a c e s y e l l o w and henna t o dye f e e t r e d i n I n d i a . C h i n e s e used v e g e t a b l e d y e s on f e e t , c h e e k s , and t i p s o f t h e i r t o n g u e s f o r v a r i o u s r e a s o n s . The Romans u s e d w h i t e l e a d and c h a l k o n t h e i r f a c e s and b l u e and g o l d dye o n t h e i r h a i r . The s o u r c e s o f d y e s u s e d b y man i n c l u d e a n i m a l , v e g e t a b l e , and m i n e r a l . S i r W i l l i a m H e n r y P e r k i n s , i n 1856, synthesized t h e f i r s t a n i l i n e dye. I n I 8 6 0 , a t r i p h e n y l m e t h a n e dye, f u c h s i n e , was u s e d b y t h e F r e n c h t o c o l o r w i n e . On A u g u s t 2, 1886, t h e U.S. C o n g r e s s a u t h o r i z e d t h e a d d i t i o n o f c o l o r t o b u t t e r . On J u n e 6, 1896, C o n g r e s s a p p r o v e d c o l o r a n t s i n c h e e s e , a n d b y 1900 c o l o r a n t s were added t o c a t s u p , j e l l i e s , c o r d i a l s , c a n d i e s , sausage and noodles. However, t h e r e were some c o n c e r n s b y t h e p u b l i c . F o r example, chrome y e l l o w , m a r t i u s y e l l o w a n d q u i c k s i l v e r v e r m i l l i o n were added t o f o o d s t o h i d e p o o r q u a l i t y o r t o i n c r e a s e w e i g h t . T h e r e was no c o n t r o l o v e r t h e p u r i t y o f c o l o r a n t s u s e d . F o r e x a m p l e , i t h a s been n o t e d t h a t r e j e c t e d t e x t i l e d y e s were sometimes added t o f o o d s . Use o f a r s e n i c a c i d and m e r c u r y i n t h e m a n u f a c t u r e o f c o l o r a n t s a l s o c r e a t e d some concerns. In 1899, t h e N a t i o n a l C o n f e c t i o n e r s A s s o c i a t i o n p u b l i s h e d a l i s t o f u n f i t c o l o r a n t s f o r f o o d s . T h i s was t h e f i r s t t i m e t h a t any g r o u p a d d r e s s e d t h e i s s u e o f s a f e t y o f c o l o r a n t s . Unfort u n a t e l y , t h i s was n o t v e r y e f f e c t i v e . I n A u g u s t , 1904, t h e U.S.D.A. i s s u e d a F o o d I n s p e c t i o n D e c i s i o n w h i c h s t a t e d , "Food i s a d u l t e r a t e d i f i t i s c o l o r e d , powdered o r p o l i s h e d w i t h attempt t o deceive." A n o t h e r F o o d I n s p e c t i o n D e c i s i o n was i s s u e d i n September 1905 i n w h i c h t h e U.S.D.A. r e q u i r e d t h a t c o l o r s must b e d e c l a r e d on l a b e l s . I n 1906 t h e U.S.D.A. d e c l a r e d m a r t i u s y e l l o w i n m a c a r o n i t o be an u n s a f e c o l o r ; a n d i n 1907 t h e F.D.A. p r o h i b i t e d t h e u s e o f d a n g e r o u s and impure c o l o r a n t s i n f o o d . T h e Food a n d Drug A c t was p a s s e d i n 1906. Dr. B e r n a r d C. H e s s e o f t h e U.S. D e p a r t m e n t o f A g r i c u l t u r e had e v a l u a t e d t h e c h e m i s t r y and p h y s i o l o g y o f a p p r o x i m a t e l y 700 c o a l t a r d y e s . O n l y c o l o r s o f known c o m p o s i t i o n were examined p h y s i o l o g i c a l l y . T h o s e s h o w i n g n o n - f a v o r a b l e r e s u l t s c o u l d be u s e d i n f o o d s . The U.S.D.A. began t o c e r t i f y s y n t h e t i c o r g a n i c f o o d c o l o r s i n A p r i l 1908. T h e c o s t o f t h e c e r t i f i c a t i o n was t o b e b o r n e b y i n d u s t r y (and s t i l l i s ) . T h e c o l o r s a p p r o v e d a t t h a t t i m e were amaranth (Red 2 ) , p o n c e a u 3R ( O r a n g e 1 ) , e r y t h r o s i n e (Red 3 ) , n a p h t h o l y e l l o w S ( Y e l l o w 7 ) , l i g h t g r e e n SF y e l l o w i s h , and i n d i g o d i s u l f o a c i d s o d i u m s a l t ( B l u e 2 ) . O t h e r c o l o r s were added: i n 1916, t a r t r a z i n e ( Y e l l o w 5 ) ; i n 1927, FCF g r e e n

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch020

20.

BORZELLECA ET AL.

Food,

Drug,

and Cosmetic

Colors

313

G r e e n 3 ) ; a n d 1 9 2 9 p o n c e a u SX ( R e d 4 ) , s u n s e t y e l l o w SCF Y e l l o w 6 ) , a n d b r i l l i a n t b l u e SCF ( B l u e 1 ) . I n 1938, t h e F e d e r a l F o o d , Drug and C o s m e t i c A c t was p a s s e d . I t s t a t e d t h a t u n c e r t i f i e d c o a l t a r c o l o r s i n food, drugs and cosmetics could n o t b e s h i p p e d i n i n t e r s t a t e commerce. U s e o f c o l o r s was l i m i t e d . A l l c o l o r s h a d t o be c e r t i f i e d a n d h a r m l e s s . T h r e e c a t e g o r i e s o f c o a l t a r c o l o r s were e s t a b l i s h e d : F o o d , Drug a n d C o s m e t i c c o l o r s , Drug and C o s m e t i c c o l o r s , a n d E x t e r n a l Drug and C o s m e t i c c o l o r s . T h e S e r v i c e and R e g u l a t o r y Announcement, FD and C No. 3, September 1940, l i s t e d c o l o r s t h a t were a p p r o v e d a t t h a t time and t h e s p e c i f i c a t i o n and r e g u l a t i o n s concerning manufacturing, c e r t i f i c a t i o n , sale, etc. T h e F o o d and Drug A d m i n i s t r a t i o n began t e s t i n g c o l o r s i n t h e e a r l y 1950s f o l l o w i n g an i n c i d e n t i n w h i c h c h i l d r e n became s i c k a f t e r e a t i n g c a n d y and colored popcorn. Unfavorable f i n d i n g s r e s u l t e d i n the banning o f FD a n d C Orange 1, FD and C Orange 2, and FD and C Red 3 2 . The FDA's p o s i t i o n was t h a t t h e t e r m h a r m l e s s n e s s a s d e f i n e d i n t h e 1938 A c t meant s a f e r e g a r d l e s s o f t h e amount u s e d . Later, t h e Supreme C o u r t r u l e d t h a t t h e F o o d a n d Drug A d m i n i s t r a t i o n could n o t e s t a b l i s h l i m i t s unless t h e c e r t i f i e d c o l o r i n any q u a n t i t y c a u s e d harm. T h e C o l o r A d d i t i v e s Amendment o f 1960 (PL-86-618) r e q u i r e d t h e f o l l o w i n g : e x i s t i n g c o l o r s c o u l d be u s e d p e n d i n g f u r t h e r t e s t i n g ; l i m i t s o f u s e were e s t a b l i s h e d ; a l l c o l o r s were t o be i n c l u d e d , n o t j u s t t h e c o a l t a r c o l o r s ; t h e S e c r e t a r y was t o d e t e r m i n e w h i c h c o l o r s were t o be c e r t i f i e d and w h i c h w o u l d b e exempted; p r o d u c e r s and c o n s u m e r s were t o p r o v i d e d a t a t o o b t a i n permanent l i s t i n g s ; and p r o v i s i o n a l l i s t i n g r e f e r r e d t o c o l o r s f o r w h i c h a d d i t i o n a l d a t a were n e c e s s a r y t o s e c u r e permanent l i s t i n g . Uses o f C o l o r s F o o d s . C o l o r s a r e added ( 1 ) t o f o o d s t h a t have no c o l o r o f t h e i r own ( b e v e r a g e s , g e l a t i n d e s s e r t , c a n d i e s , i c e c r e a m ) ; (2) where t h e n a t u r a l c o l o r h a s been l o s t i n p r o c e s s i n g o r s t o r a g e ; (3) where c o l o r o f t h e f o o d v a r i e s w i t h t h e s e a s o n o f the y e a r (geographic o r i g i n , f o r example d a i r y products, oranges); ( 4 ) t o make f o o d s r e c o g n i z a b l e and a t t r a c t i v e , e n h a n c i n g t h e i r a e s t h e t i c v a l u e . T h e meat i n s p e c t i o n stamp i s also colored. D r u g s . C o l o r s a r e added t o d r u g s f o r p u r p o s e s o f i d e n t i f i c a t i o n , s t a n d a r d i z i n g b a t c h e s , t o mask u n s a t i s f a c t o r y n a t u r a l c o l o r s , and f o r a p p e a l ( c o l o r l e s s p r o d u c t s a r e not a e s t h e t i c ) . Cosmetics. Cosmetics r e q u i r e the a d d i t i o n o f c o l o r s f o r e f f e c t i v e n e s s . C o l o r s may b e added t o e n h a n c e t h e a e s t h e t i c v a l u e , f o r example t o a f t e r s h a v e l o t i o n a n d shampoo, o r t o s e r v e a f u n c t i o n a l p u r p o s e , f o r example i n eye brow p e n c i l , n a i l p o l i s h ,

314

X E N O B I O T I C S IN F O O D S A N D

l i p s t i c k , e t c . T h e amounts u s e d i n c o s m e t i c s i s used i n f o o d s and d r u g s . Acceptable

i s greater

FEEDS

than

D a i l y Intake (API)

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch020

The amount o f t h e m a t e r i a l t h a t c a n be i n g e s t e d w i t h o u t u n r e a s o n a b l e r i s k o f a d v e r s e h e a l t h e f f e c t s may be c o n s i d e r e d an a c c e p t a b l e d a i l y i n t a k e . This r e f e r s p r i m a r i l y t o food c h e m i c a l s . T o a r r i v e a t an a c c e p t a b l e d a i l y i n t a k e , a p p r o p r i a t e t o x i c o l o g i c a l t e s t s a n d s a f e t y f a c t o r s a r e i n v o l v e d . ADIs h a v e been e s t a b l i s h e d b y t h e U.S. F o o d a n d Drug A d m i n i s t r a t i o n , t h e W o r l d H e a l t h O r g a n i z a t i o n , t h e J o i n t E x p e r t Committee o n F o o d A d d i t i v e s , and t h e E u r o p e a n E c o n o m i c Community. B a c k g r o u n d o f t h e C u r r e n t T e s t i n g Program The Food and Drug A d m i n i s t r a t i o n d e t e r m i n e d t h a t t h e p r o v i s i o n a l l i s t i n g o f c o l o r s was t o be e l i m i n a t e d . C o l o r s were t o be e i t h e r p e r m a n e n t l y l i s t e d o r t o b e p r o h i b i t e d f r o m u s e . A l s o , t h e r e was c o n c e r n a b o u t c o l o r s t h a t were p e r m a n e n t l y l i s t e d . The c o n c e r n was n o t b a s e d o n a d v e r s e h e a l t h e f f e c t s n o r on t h e g e n e r a t i o n o f a s i g n i f i c a n t amount o f t o x i c o l o g i c a l d a t a , but p r e s u m a b l y o n a t e r a t o l o g y s t u d y o f Aramanth (Red No. 2) t h a t a p p e a r e d i n R u s s i a n l i t e r a t u r e . The f i n d i n g s were n o t c o n f i r m e d when t h e s t u d y was r e d o n e b y t h e Food and Drug A d m i n i s t r a t i o n and by t h e C e r t i f i e d C o l o r s Manufacturers A s s o c i a t i o n . T h e r e was some c o n c e r n t h a t c o l o r s were n o t n e c e s s a r y s i n c e t h e y are non-nutrive. This neglects t h e aesthetic value o f foods. C o l o r s were a l s o r e - e v a l u a t e d , p r e s u m a b l y b e c a u s e o f t h e c y c l i c r e v i e w a n d r e - e v a l u a t i o n p r o g r a m a t FDA. T h e i n d u s t r y was r e q u e s t e d b y FDA t o r e - t e s t a l l FD a n d C and D and C c o l o r s . S p e c i f i c a l l y , t h e c o l o r m a n u f a c t u r e r s were r e q u e s t e d t o p r o v e s a f e t y . I f s a f e t y i s d e f i n e d as t h e absence o f t o x i c i t y , then i t can never be proved s i n c e one cannot prove a n e g a t i v e . I f s a f e t y i s d e f i n e d a s t h e v e r y l o w p r o b a b i l i t y t h a t an a d v e r s e e f f e c t w i l l o c c u r under c e r t a i n c o n d i t i o n s o f u s e , t h e n a p p r o p r i a t e s t u d i e s c a n b e d e s i g n e d and l e v e l s d e t e r m i n e d . H a r m l e s s n e s s must b e d e f i n e d under c e r t a i n c o n d i t i o n s o f u s e . I n o r d e r t o r e s p o n d t o FDA's r e q u e s t , l i f e - t i m e s t u d i e s were c o n d u c t e d i n t h e mouse a n d t h e r a t t o e s t a b l i s h s a f e t y ( r e a s o n a b l e c e r t a i n t y t h a t no harm w i l l o c c u r when t h e m a t e r i a l i s used under certain conditions). P r i o r t o i n i t i a t i n g these s t u d i e s , a thorough search o f the l i t e r a t u r e was c o n d u c t e d a n d t h e e x t e n t o f o r a l t o x i c i t y t e s t s was d e t e r m i n e d . I t was f o u n d t h a t t h e c o l o r s have a v e r y l o w o r d e r o f a c u t e o r a l t o x i c i t y . An e x p e r i m e n t a l d e s i g n was t h e n developed. P o t e n t i a l r e p r o d u c t i v e t o x i c i t i e s were e v a l u a t e d i n multigeneration r e p r o d u c t i v e and t e r a t o l o g i c a l s t u d i e s . No a d v e r s e e f f e c t s were r e p o r t e d a t any o f t h e l e v e l s e x a m i n e d .

20.

BORZELLECA ET

Experimental

AL.

Food,

Drug,

and Cosmetic

Colors

315

Design

The FD and C c o l o r s e v a l u a t e d a r e l i s t e d i n Table I. The c h e m i c a l s t r u c t u r e s , m o l e c u l a r w e i g h t s and c h e m i c a l names a p p e a r i n Table i i . The suppliers o f t h e colors a r e i d e n t i f i e d i n Table i n . O t h e r e x p e r i m e n t a l d e t a i l s a r e summarized i n Tables i v

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch020

through V I I .

The c h r o n i c t o x i c i t y / c a r c i n o g e n i c i t y s t u d i e s were d e s i g n e d as d e f i n i t i v e s t u d i e s t o a d d r e s s t h e i s s u e o f s a f e t y . T h e e x p e r i m e n t a l d e s i g n i n v o l v e d j_n u t e r o e x p o s u r e o f r a t s and t h e use o f w e a n l i n g m i c e . O n l y c e r t i f i e d b a t c h e s o f c o l o r f r o m t h e v a r i o u s s u p p l i e r s were u s e d . Two l a b o r a t o r i e s were s e l e c t e d f o l l o w i n g an e x t e n s i v e e v a l u a t i o n o f t h e f a c i l i t i e s a v a i l a b l e at that time. The c o l o r s were a n a l y z e d on a r e g u l a r b a s i s t o e v a l u a t e s t a b i l i t y , and m i c r o b i o l o g i c a l contamination. Feed analyses were c o n d u c t e d o n a w e e k l y b a s i s f o r t h e f i r s t 13 weeks o f t h e s t u d y , and t h e n a t m o n t h l y i n t e r v a l s t h e r e a f t e r b y t h e c o n t r a c t l a b o r a t o r y a n d b y i n d e p e n d e n t l a b o r a t o r i e s . Good l a b o r a t o r y p r a c t i c e s monitoring occurred a t l e a s t monthly. Results The r e s u l t s o f t h e s e s t u d i e s a r e summarized i n T a b l e s V I I I through XIII. These data f a i l e d t o i d e n t i f y any carcinogenic p o t e n t i a l o f t h e F o o d , Drug and C o s m e t i c c o l o r s . T h e r e were no c o n s i s t e n t , b i o l o g i c a l l y s i g n i f i c a n t compound-related e f f e c t s a t any l e v e l in e i t h e r species. Future

Studies

The g e n e t i c t o x i c o l o g i c a l a s p e c t s o f t h e c o l o r s a r e b e i n g evaluated. The data generated t o date f a i l e d t o demonstrate g e n o t o x i c i t y o f t h e s i x F D and C c o l o r s e v a l u a t e d . Comparative b i o t r a n s f o r m a t i o n and k i n e t i c s t u d i e s a r e e s s e n t i a l and t h e s e are i n p r o g r e s s . Special studies are being considered i n c l u d i n g i n t e r a c t i o n s , s e n s i t i z a t i o n o r a l l e r g i e s , and a s s e s s m e n t o f t h e v a l u e o f c o l o r i n human n u t r i t i o n . Summary and

Conclusions

The F o o d , Drug a n d C o s m e t i c c o l o r s h a v e been e x t e n s i v e l y e v a l u a t e d ( f e w f o o d c h e m i c a l s have been s o e x t e n s i v e l y s t u d i e d ) . The F o o d , Drug and C o s m e t i c c o l o r s d o n o t p o s e a t h r e a t t o human health a t l e v e l s c u r r e n t l y i n use o r a t l e v e l s greater than t h o s e c u r r e n t l y u s e d . T h e r e i s no b i o l o g i c a l e v i d e n c e i n a n i m a l s o r human t h a t t h e F o o d , Drug and C o s m e t i c c o l o r s a r e u n s a f e o r h a z a r d o u s t o human h e a l t h .

FD and C B l u e No. 1 FC and C G r e e n No. 3 FD and C B l u e No. 2 FD and C Y e l l o w No. 6 FD and C Y e l l o w No. 5 FD and C Red No. 3

T r i phenylmethane:

Indigoid:

Monoazo:

Pyrazolone:

Xanthene:

Chemical C l a s s

(Erythrosine) (Tetraiodo Fluorescein)

(Tartrazine)

(Sunset Yellow FCF)

(Indigotine)

1907

1916

1929

1907

1929 1927

Year approved by FDA/USDA ( B r i l l i a n t Blue FCF) ( F a s t Green FCF)

Chronic Toxicity Studies 1977-1981

FD AND C COLORS

Table I

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch020

20.

BORZELLECA E T AL.

Table

II.

Food,

and Cosmetic

x

Empirical

FD&C Red Red No. No. 40 FD&C 40 ( A l l u r a Red AC) FD&C Red No. : 3 (Erythrosine)

H

N

Formula

C

18 14°8 2 2

C

20 6 4

H

I

N a

S

N a

2

2°5

FD&C Y e l l o w No. 5

n

Q

Weights

M o l e c u l a r Weijght

496.40

9 1 2

C,,H N.Na 0 S 16 9 4 3 9 2

\

317

879.87

VeWs frr,

Colors

Çhem i ç a^l _S t^uc^jur e s . Name sj,. _ a n d __Mo 1 e çu l a r o f t h e FD&C C o l o r s E v a l u a t e d

Color

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch020

Drug,

Q

o

·

1 0

534.37

(Tartrazme) FD&C YYeellllooww No. 6 (Sunset Y e l l o w FCF) FD&C B l u e No. No 2 (Indigotine) FD&C G re ee en n Ν FD&C Gr No. 3 ( F a s t Green FCF)

C

16 10 2

H

C

16 8 2

C

37 34 2

H

H

N

N

N

N a

N a

S

2°7 2

S

2°8 2

N a

452.37

466.36

S

2°10 3

Continued

808.86

on n e x t

page

318

X E N O B I O T I C S IN F O O D S A N D

Table

II.

Chemical

S t r u c t u r e s ,

Color

Dye

FD&C Red No. 40 ( A l l u r a Red AC)

Monoazo

FD&C

Xanthene

Red

No.

3

Names,

Type

and

Molecular

Weights

Structure

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch020

(Erythrosine)

FD&C

Yellow

No.

5

Pyrazolone

6

Monoazo

(Tartrazine)

FD&C

Yellow

(Sunset

FD&C

No.

Yellow

Blue

FCF)

2

No.

Indigoid

(Indigotine) H

FD&C (Fast

Green Green

No.

3

FCF)

Triphenylmethane

FEEDS

0

20.

of

BORZELLECA ETAL.

Drug,

t h e FD&C C o l o r s E v a l u a t e d —

Chemical

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch020

Food,

and Cosmetic

Colors

Continued

CFR

Name

Reference

Disodium s a l t of 6-hydroxy-5[(2-methoxy-5-methyl-4-sulfophenyl)azo]-2-napththalenesulfonic acid

21 CFR

74.340

Monohydrate of 9-(0-carboxyphenyl)-6-hydroxy-2,4,5,7-tetraiodo3H-xanthen-3-one, d i s o d i u m o r d i p o t a s s i u m s a l t , w i t h s i m i l a r amounts o f l o w e r iodinated fluoresceins

21 CFR

74.303

5-0xo-l-(p-sulfophenyl)-4-[(p-sulfophenyl)azo]-2-pyrazoline-3-carboxy1ic acid, trisodium salt

21 CFR

74.705

Disodium s a l t of 1-p-sulfophenylazo-2-naphthol-6-sulfonic acid

21 CFR

82.706

P r i n c i p a l l y the disodium s a l t , o f 5,5'-disulfo-3,3'-dioxo- Δ ' b i i n d o l i n e w i t h s m a l l e r amounts o f the i s o m e r i c disodium^sg].t o f 5,7 disulfo-3,3'-dioxo-Δ ' -biindoline

21 CFR

74.1102

4-[(4-Εthy1-p-sulfobenzylamino)phenyl]-(4-hydroxy-2-sulfoniumphenyl)methylene^-[l-(N-ethyl-N-p-sulfobenzyl)-A ' -cyclohexadienimine]

21 CFR

82.203

f

X E N O B I O T I C S IN F O O D S A N D

FEEDS

Table III FD AND C COLORS

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch020

Chronic T o x i c i t y Studies 1977-1981

SUPPLIERS OF COLORS Color

Manufacturer

FD and C B l u e No. 1

H i l t o n - D a v i s Chemical Co.

FD and C G r e e n No. 3

Warner-Jenkinson Co.

FD and C B l u e No. 2

H i l t o n - D a v i s Chemical Co.

FD and C Y e l l o w No. 6

Stange Co. H i l t o n - D a v i s Chemical Co.

FD and C Y e l l o w No. 5 Warner-Jenkinson Co. FD and C Red No. 3 H. Kohnstamm

Rat Mouse

International Research and Development C o r p .

P u r i n a Rodent L a b o r a t o r y Chow P u r i n a Rodent L a b o r a t o r y Chow A f t e r 8/79: Purina Certified L a b o r a t o r y Chow

R a t and Mouse R a t and Mouse

International Research and Development C o r p .

Feed

P o r t a g e , MI

C h a r l e s R i v e r CD R a t s C h a r l e s R i v e r CD-I M i c e

Species

W i l m i n g t o n , MA

Colony

C h a r l e s R i v e r CD R a t s C h a r l e s R i v e r CD-I M i c e

Strain

Biodynamics

Laboratory

ANIMAL FEED

Rat Mouse

Biodynamics

MATERIALS AND METHODS:

Species

ANIMALS

Laboratory

MATERIALS AND METHODS:

Chronic Toxicity Studies 1977-1981

FD AND C COLORS

T a b l e IV

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch020

X E N O B I O T I C S IN F O O D S A N D

322

FEEDS

Table V FD AND C COLORS Chronic T o x i c i t y Studies 1977-1981 MATERIALS AND METHODS:

MICE

( C h a r l e s R i v e r , CD-I)

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch020

Observation General changes, moribundity, m o r t a l i t y (3 χ d a i l y ) Body W e i g h t / F o o d C o n s u m p t i o n ( w e e k l y : weeks 1-14; b i - w e e k l y : weeks 16-26 e v e r y 4 weeks t h e r e a f t e r ) Detailed Physical Examination ( w e e k l y : weeks 1-14; b i - w e e k l y : weeks 16-16; e v e r y 4 weeks t h e r e a f t e r )

Pathology Gross pathology on a l l animals DOS a n i m a l s Terminal

Sacrifice

Statistical Analysis Appropriate tests

Test Diet Analysis Independent a n a l y s i s by c o n t r a c t l a b and CCMA member company l a l

C l i n i c a l Laboratory Tests H e m a t o l o g y ( 3 , 6, 12, 18 months; terminal) Hemoglobin Hematocrit Total erythrocyte E r y t h r o c y t e morphology T o t a l and d i f f e r e n t i a l l e u c o c y t e (10 Μ , 10 F f r o m each g r o u p )

Histopathology Adrenal (2) A o r t a (abdominal) Bone and bone marrow ( f e m u r ) B l o o d smear B r a i n (3 s e c t i o n s , i n c l u d i n g f r o n t a l c o r t e x and b a s a l g a n g l i a , p a r i e t a l c o r t e x and t h a l a m u s ; c e r e b e l l u m and p o n s ) Esophagus Eye ( 2 - w i t h o p t i c n e r v e ) Gall bladder Heart (with coronary v e s s e l s ) Intestine cecum colon duodenum ileum Kidneys (2) Liver (2) Lung and m a i n s t e m b r o n c h i Lymph nodes (mesenteric, mediastinal)

BORZELLECA ET AL.

Food,

Table V

Drug,

and Cosmetic

Colors

(Continued)

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch020

Histopathology Mammary g l a n d ( i n g u i n a l ) Nerve ( s c i a t i c ) Ovaries Pancreas Pituitary Prostate S a l i v a r y gland (mandibular) S e m i n a l v e s i c l e s (2) S k e l e t a l muscle (biceps femoris) Skin Spinal cord ( c e r v i c a l ) Spleen Stomach Testes with epididymides Thymus Thyroid/parathyroid Trachea Urinary bladder Uterus Gross changes o r u n c e r t a i n nature (including a s e c t i o n o f normala p p e a r i n g p o r t i o n o f same tissue) T i s s u e masses o r s u s p e c t tumors w i t h r e g i o n a l lymph nodes

X E N O B I O T I C S IN F O O D S A N D

324

FEEDS

T a b l e VI FD AND C COLORS Chronic T o x i c i t y Studies 1977-1981 MATERIALS AND METHODS:

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch020

In U t e r o

RATS

Segment

Group

Male

Female

M i n . No. L i t t e r s

Dosage L e v e l s

1-A

60

60

70

Control

1-B

60

60

70

Control

2

60

60

35

Low

3

60

60

35

Mid

4

60

60

35

High

RANDOM SELECTION

Chronic Feeding

Segment

Group

Male

Female

Dosage L e v e l s

1-A

70

70

Control

1-B

70

70

Control

2

70

70

Low

3

70

70

Mid

4

70

70

High

20.

BORZELLECA ET AL.

Food,

Drug,

and Cosmetic

Colors

325

Table VII FD AND C COLORS Chronic T o x i c i t y Studies 1977-1981 MATERIALS AND METHODS:

RATS ( C h a r l e s R i v e r , CD-I)

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch020

Observation General changes, moribundity,) m o r t a l i t y (3 χ d a i l y ) Body W e i g h t / F o o d C o n s u m p t i o n ( w e e k l y : weeks 1-14; b i - w e e k l y : weeks 16-26 e v e r y 4 weeks t h e r e a f t e r ) Detailed Physical Examination ( w e e k l y : weeks 1-14; b i - w e e k l y : weeks 16-16; e v e r y 4 weeks t h e r e a f t e r ) Pathology Gross pathology on a l l animals DOS a n i m a l s Interim S a c r i f i c e Terminal S a c r i f i c e

C l i n i c a l Laboratory

Tests

H e m a t o l o g y ( 3 , 6, 12, 1 8 , 24 months; t e r m i n a l ) Hemoglobin Hematocrit Total erythrocyte E r y t h r o c y t e morphology T o t a l and d i f f e r e n t i a l l e u c o c y t e (10 M, 10 F f r o m e a c h g r o u p ) B l o o d C h e m i s t r y ( 3 , 6, 1 2 , 18, 24 months; t e r m i n a l ) Serum g l u t a m i c o x a l o a c e t i c transaminase Serum g l u t a m i c p y r u v i c transaminase A l k a l i n e phosphatase Blood urea nitrogen Fasting glucose Total protein Creatinine

U r i n a l y s i s ( 3 , 6, 1 2 , 18 24 months; t e r m i n a l ) Gross appearance Appropriate tests Specific gravity pH Protein Test Diet Analysis Glucose Ketones Independent a n a l y s i s by c o n t r a c t Bilirubin l a b and CCMA member company l a i O c c u l t b l o o d Microscopic analysis Statistical Analysis

(Continued)

326

X E N O B I O T I C S IN F O O D S A N D

T a b l e VII

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch020

Histopathology Adrenal (2) Aorta (abdominal) B l o o d smear Bone and bone marrow ( f e m u r ) B r a i n (3 s e c t i o n s , i n c l u d i n g f r o n t a l c o r t e x and b a s a l g a n g l i a , p a r i e t a l c o r t e x and t h a l a m u s ; c e r e b e l l u m and pons) Esophagus E y e (2 - w i t h o p t i c n e r v e ) Heart (with coronary v e s s e l s Intestine cecum colon duodenum ileum Kidneys (2) Liver (2) Lung and m a i n s t e m b r o n c h i Lymph nodes (mesenteric, mediastinal) Mammary g l a n d ( i n g u i n a l ) Nerve ( s c i a t i c ) Ovaries Pancreas Pituitary Prostate S a l i v a r y gland (mandibular) Seminal v e s i c l e s ( 2 ) S k e l e t a l muscle (biceps femoris) Skin Spinal cord ( c e r v i c a l ) Spleen Stomach Testes with epididymides Thymus Thyro i d/parathyro i d Trachea Urinary bladder Uterus Gross changes o f u n c e r t a i n nature (including a s e c t i o n o f normala p p e a r i n g p o r t i o n o f same t i s s u e ) T i s s u e masses o r s u s p e c t tumors w i t h r e g i o n a l lymph nodes

FEEDS

(Continued) Ophthalmoscopic

Examination

3, 6, 12, 18, 24 months

Controls

2 Concurrent Controls

5.0% (7500 mg/kg/day)

1.5% (2250 mg/kg/day)

0 . 5 % (750 mg/kg/day)

60/Sex/Group

2 Concurrent

2.0% (1000 mg/kg/day)

1.0% (500 mg/kg/day)

0 . 1 % (50 mg/kg/day)

70/Sex/Group

Number/Group Dose L e v e l s

F e m a l e s - 2 8 — 3 0 A p r i l 1980

M a l e s - 28-30 A p r i l 1978

Termination:

26 A p r i l 1978

Initiation:

F e m a l e s - 15 November 1979

M a l e s - 28 December 1979

Termination:

11 O c t o b e r 1977

Initiation:

Initiation Termination

No c o n s i s t e n t biologically significant compound r e l a t e d adverse e f f e c t s

No c o n s i s t e n t biologically significant compound r e l a t e d adverse e f f e c t s

Effects

9

ρ

to

a.

is

fx to S

ο

?

>

^

Co

S"

Cosm etic d

Mouse

Rat

Species

FD AND C BLUE NO. 1

I n t e r n a t i o n a l R e s e a r c h and D e v e l o p m e n t C o r p o r a t i o n

Table VIII

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch020

ELLECA ET

Mouse

Rat

Species

2 Concurrent Controls

5.σ/ο (7500 mg/kg/day)

16 May 1980

Termination:

11 May 1978

0.5% (750 mg/kg/day)

1.5% (2250 mg/kg/day)

Initiation:

F e m a l e s - 18 A p r i l 1980

M a l e s - 4 March 1980

Termination:

60/Sex/Group

2 Concurrent Controls

5.0% (2500 mg/kg/day)

2.5% (1250 mg/kg/day)

12 O c t o b e r 1977

Initiation:

Initiation Termination Effects

No c o n s i s t e n t biologically significant compound r e l a t e d adverse e f f e c t s

Statistically significant increase i n hyperplasia and t u m o r s i n u r i n a r y b l a d d e r s o f high-dose male rats.

FD AND C GREEN NO. 3 B i o / D y n a m i c s , I n c .

1.25% (625 mg/kg/day)

70/Sex/Group

Number/Group Dose L e v e l s

T a b l e IX

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch020

2 Concurrent C o n t r o l s

5.054 (7500 mg/kg/day)

1.554 (2250 mg/kg/day)

Males - 21-22 February 1980

Termination:

No c o n s i s t e n t biologically significant compound r e l a t e d adverse e f f e c t s

*Not b i o l o g i c a l l y significant

Females - 3-4 A p r i l 1980

l b May 1978

Mammary Tumors (Males)*

Males - 18-19 February 1980

0.554 (750 mg/kg/day)

Bladder Tumors (Males)*

Termination:

Initiation:

B r a i n Gliomas and Granular C e l l Tumors (Males)*

Effects

18 October 1977

Initiation:

60/Sex/Group

2 Concurrent C o n t r o l s

2.σ/ο (1000 mg/kg/day)

1.0% (500 mg/kg/day)

0.5% (250 mg/kg/day)

70/Sex/Group

Initiation Termination

Females - 14-15 A p r i l 1980 * S t a t i s t i c a l l y s i g n i f i c a n t with high dose males only

Mouse

Rat

Species

Number/Group Dose L e v e l s

Bio/Dynamics, Inc.

FD AND C BLUE NO. 2

Table Χ

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch020

Κ)

s

o

o

> r

H

r r m n >

Ν M

*>

Ο

Ο

Mouse

Rat

Species

2 Concurrent Controls

5.σ/ο (7500 mg/kg/day)

Females - 30 March 1980

Males - 29 December 1979

Termination:

16 May 1978

0.5% (750 mg/kg/day)

1.5/o (2250 mg/kg/day)

Initiation:

60/Sex/Group

5.σ/ο - March 1981

Females - 7 March 1980

Males - 22 A p r i l 1980

5.σ/ο (2500 mg/kg/day) Controls

Termination:

3.σ/ο (1500 mg/kg/day)

2 Concurrent

5.σ/ο - 26 February 1979

3 November 1977

Initiation:

Initiation Termination

1.5/o (750 mg/kg/day)

0.75/o (375 mg/kg/day)

70/Sex/Group

Number/Group Dose Levels

Bio/Dynamics, Inc.

FD AND C YELLOW NO. 6

Table XI

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch020

No c o n s i s t e n t biologically significant compound r e l a t e d adverse e f f e c t s

No c o n s i s t e n t biologically significant compound r e l a t e d adverse e f f e c t s

Effects

(Λ

Ό

m m

τ]

> Ζ σ

Ό

τι Ο Ο

η

Η

X m ζ ο 2 δ

°

Mouse

Rat

Species

Termination:

2.0% (1000 mg/kg/day)

2 Concurrent Controls

5.0% (7500 mg/kg/day)

2 May 1980

Termination:

3 May 1978

0.5% (750 mg/kg/day)

1.5% (2250 mg/kg/day)

Initiation:

5.0» - 24 F e b r u a r y 1981

60/Sex/Group

2 Concurrent Controls

5.0% - 9 O c t o b e r 1978

1.0% (500 mg/kg/day)

31 December 1980

25 O c t o b e r 1977

Initiation:

Initiation Termination

0 . 1 % (50 mg/kg/day)

70/Sex/Group

Number/Group Dose L e v e l s

Table XII FD AND C YELLOW NO. 5 I n t e r n a t i o n a l R e s e a r c h and D e v e l o p m e n t C o r p o r a t i o n

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch020

No c o n s i s t e n t biologically significant compound r e l a t e d adverse e f f e c t s

No c o n s i s t e n t biologically significant compound r e l a t e d adverse e f f e c t s

Effects

Mouse

Rat

Species

2 Concurrent

Controls

3.054 (4500 mg/kg/day)

1.054 (1500 mg/kg/day)

0.3% (500 mg/kg/day)

60/Sex/Group

Controls

(50 mg/kg/day) (250 mg/kg/day) (500 mg/kg/day) (2000 mg/kg/day)

2 Concurrent

0.1% 0.5% 1.054 4.054

70/Sex/Group

Number/Group Dose L e v e l s

I n t e r n a t i o n a l Research

Table XIII

23 A p r i l 1980

Termination:

25 A p r i l 1978

Initiation:

27 March 1980 4% - 24 F e b r u a r y 1981

Termination:

18 O c t o b e r 1977 4% - 4 O c t o b e r 1978

Initiation:

Initiation Termination

and D e v e l o p m e n t C o r p o r a t i o n

FD AND C RED NO. 3

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch020

No c o n s i s t e n t biologically significant compound r e l a t e d adverse e f f e c t s

No c o n s i s t e n t biologically significant compound r e l a t e d adverse e f f e c t s

Effects

21 Analysis of Carrot Constituents: Myristicin, Falcarinol, and Falcarindiol S. G. YATES, R. E. ENGLAND, and W. F. KWOLEK

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch021

Northern Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, Peoria, IL 61604 P. W. SIMON Department of Horticulture, University of Wisconsin, Madison, WI 53706 Falcarinol, falcarindiol, and myristicin contents of carrots, Daucus carota L . , were determined by a sequence of dichloromethane extraction, column chromatographic purification, and gas-liquid chromatographic analysis. High Color 9, Long Imperator 58, Danvers 126, and Spartan Bonus varieties were grown in Wisconsin (1979-1982), Florida (1980-1982), California (1980-1982), Arizona (1981), and Illinois (1981-1982). Gold Pak, Nantes Half Long, Red Cored Chantenay, and Royal Chantenay varieties were grown in Illinois (1980). The overall mean of falcarinol for 510 observations of these eight commercial varieties was 24.1 mg/kg; that of falcarindiol for 389 observations was 65.1 mg/kg. The standard error of a mean based on 2 samples of 4 carrots, with 2 aliquots per sample, was 2.8 for falcarinol and 4.8 for falcarindiol. Varietal means ranged from 11.3 to 28.2 for falcarinol and 53.3 to 106.9 for falcarindiol. Myristicin was detected in only one variety of carrots (Spartan Bonus) harvested in Wisconsin in 1981; the mean of 12 observations, 2 samples, was 1.4 mg/kg with a range of 1.3 to 1.5 mg/kg. This chapter not subject to U.S. copyright. Published 1983, American Chemical Society

X E N O B I O T I C S IN F O O D S A N D F E E D S

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch021

334

Crosby and Aharonson ( 1 ) , i n the course o f t h e i r i n v e s t i g a t i o n o f n a t u r a l l y o c c u r r i n g t o x i c a n t s i n foods, discovered that an acetone e x t r a c t of c a r r o t s was t o x i c t o the organism Daphnia magna Straus. The p u r i f i e d t o x i n had an L D ^ Q i n mice of 100 mg/kg. They gave t h i s substance the t r i v i a l name " c a r o t a t o x i n " and published a t e n t a t i v e s t r u c t u r e . Bentley and T h a l l e r (2) published a corrected s t r u c t u r e and gave proof that the compound Crosby and Aharonson i s o l a t e d was f a l c a r i n o l , a p o l y a c e t y l e n i c a l c o h o l ( I ) (Figure 1) f i r s t i s o l a t e d by Bohlmann et a l . (3), from F a l c a r i a v u l g a r i s Bernh. Other such compounds have since been i s o l a t e d from c a r r o t s ( f a l c a r i n d i o l I I , a c e t y l f a l c a r i n d i o l I I I , and f a l c a r i n o l o n e IV) (4). Recently, the f u n c t i o n o f f a l c a r i n d i o l as an a n t i f u n g a l agent i n the disease response mechanism of c a r r o t s was recognized (5-2)· The LD , i p , i n mice of f a l c a r i n d i o l was found to be 133 mg/kg (8). M y r i s t i c i n V, a phenylpropenoid, i s frequently found i n c a r r o t s (9). I t i s known t o stimulate c e n t r a l nervous system a c t i v i t y (10) and to enhance the a c t i v i t y of c e r t a i n i n s e c t i c i d e s (11), and i t i s suspected o f being involved i n the disease response mechanism of c a r r o t s (12). Carrots were analyzed f o r f a l c a r i n o l , f a l c a r i n d i o l , and m y r i s t i c i n t o determine i f concentrations v a r i e d with respect to genetic ( v a r i e t y ) and/or environmental changes ( l o c a t i o n and y e a r ) . The data generated define the l e v e l o f those t o x i c a n t s normally present i n c a r r o t s grown f o r processing (Danvers 126, Spartan Bonus, Royal Chantenay, Red-Cored Chantenay, and Nantes H a l f Long), f o r f r e s h market (Long Imperator 58, High Color 9, and Gold Pak), and f o r genetic studies (B10138, B9304, B0493, B3615, and B10720). 5

0

Experimental S e c t i o n Plant M a t e r i a l s . Carrot r o o t s , Daucus carota L., were grown a t NRRC or a t the f o l l o w i n g l o c a t i o n s as d i r e c t e d by the Department of H o r t i c u l t u r e , U n i v e r s i t y of Wisconsin, Madison: Wisconsin, F l o r i d a , C a l i f o r n i a , and Arizona. Carrots were harvested by hand and shipped unwashed to NRRC i n p l a s t i c bags. A n a l y s i s of the four v a r i e t i e s was completed 1 t o 2 weeks a f t e r each harvest sample was received. Carrots grown i n I l l i n o i s (NRRC) were harvested by hand and analyzed immediately. Chromatography Equipment and Conditions. Gas-liquid chromatography was performed with a Bendix 2600 instrument (flame i o n i z a t i o n d e t e c t o r s ) ; i n j e c t o r temperature was 250°C and detector temperature was 270°C, with helium c a r r i e r gas a t 10-20 ml/min, a i r a t 500-600 ml/min, and hydrogen a t 50 ml/min. Columns were programmed from 80 t o 250°C a t 4°C/min with a

YATES ET A L .

Analysis

of Carrot

Constituents

R.

R,

CH =CH-C-C=C-C=C-C-CH=CH-(CH.),-CH

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch021

2

I

I

R

R

2

R

I II III IV

V

l '

2

6

cis

4

R

2

R> 3

R

4

Falcarinol

(OH, H)

(H, H)

Falcarindiol

(OH, H)

(OH, H)

Acetylfalcarindiol

(OAc, H)

(OH, H)

Falcarinolone

( =0)

(OH, H)

Myristicin

Figure

1. Structures

of carrot

constituents.

336

XENOBIOTICS

IN F O O D S A N D

FEEDS

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch021

5-min f i n a l h o l d . Glass columns 6 f t by 1/8 i n . were used, one packed with 3% SE-52 (Applied Science L a b o r a t o r i e s , Inc.) on 80-100 mesh Gas-Chrom Q (nonpolar), and one packed with 3% OV-17 (Supelco Inc.) on 100-120 mesh Chromosorb W HP (intermediate p o l a r i t y ) . Dichloromethane E x t r a c t i o n , Column Chromatographic P u r i f i c a t i o n and Gas-Liquid Chromatographic (DE-C CP-GLC) A n a l y s i s of C a r r o t s . A l o n g i t u d i n a l quarter was removed from each c a r r o t i n c l u d e d i n the sample. Each quarter was cut i n t o 2-mm cross s e c t i o n s , and these were immediately placed i n a 2 £ stainless steel blending container. Antioxidant (Antioxidant 330, E t h y l Corp. or I onox 330, S h e l l Chemicals) (2 mg/5 g c a r r o t f r e s h weight) and dichloromethane (10 ml/5 g c a r r o t ) were added q u i c k l y , and the c o n s t i t u e n t s were blended i n a commercial Waring Blendor at moderate speeds f o r a t o t a l of 12 min. A l t e r n a t e c y c l e s of blending and c o o l i n g (4 min each) reduce evaporation of dichloromethane. Any solvent losses were c o r r e c t e d f o r by weighing the capped blender and contents before and a f t e r blending and then r e p l a c i n g the l o s t solvent. Maximum blending e f f i c i e n c y was obtained with 300- to 500-g of c a r r o t s . T e f l o n s e a l s were i n s e r t e d i n the blender cup blade assembly to replace the standard s e a l s . After dichloromethane l o s t by evaporation was replaced, the mixture was blended f o r an a d d i t i o n a l 30 s and a l i q u o t s t r a n s f e r r e d to 250-ml c e n t r i f u g e b o t t l e s capped with a screw cap or Saran Wrap. The mixture was c e n t r i f u g e d f o r 15 min at 1500 g, and then 50 ml of the c l e a r e x t r a c t was removed with a l a r g e syringe f i t t e d with a long 17-gauge needle passed through the pulp. One mg of methyl palmitate as i n t e r n a l standard was added to each of two 50-ml a l i q u o t s of c e n t r i f u g e d e x t r a c t . These a l i q u o t s were concentrated under vacuum on a r o t a t i n g evaporator at 25°C to about 3 ml; 7 ml of hexane was added and the s o l u t i o n was reconcentrated to 3 ml; 4 ml of hexane was added and the s o l u t i o n was reconcentrated to 1 ml. The r e s u l t i n g hexane s o l u t i o n was chromatographed on 2 g of S i l i c a g e l (70-325 mesh; EM Reagents). Glass columns 6 X 140 mm, f i t t e d with 50-ml r e s e r v o i r s , were constructed from 8 mm o.d. glass tubing and 25 X 140 mm t e s t tubes. Yellow pigments were e l u t e d with hexane u n t i l the hexane eluate was c o l o r l e s s (40 to 60 ml). The f r a c t i o n of i n t e r e s t was e l u t e d with 20 ml of ether-hexane (45:55). The e l u a t e was concentrated to about 0.75 ml and t r a n s f e r r e d to a \ dram v i a l with t e f l o n - l i n e d cap. A n a l y s i s by GLC (3-6 μΐ i n j e c t i o n ) gave symmetrical peaks, which were integrated e l e c t r o n i c a l l y . Q u a n t i t i e s of using the formula

individual

t o x i c a n t s were c a l c u l a t e d

by

21.

YATES ET AL.

Analysis

of Carrot

mg of t o x i c a n t _! kg of sample r

Constituents

A^ W V t Ρ χ io A W V p e r a

=

F

t

337

0 3

where i s the response f a c t o r of the t o x i c a n t i n r e l a t i o n to methyl p a l m i t a t e , A and A are the areas of the t o x i c a n t and methyl palmitate gas chromatographic peaks, r e s p e c t i v e l y , W i s the quantity of methyl palmitate added i n m i l l i g r a m s , W is^the i n i t i a l weight of c a r r o t s used, V i s the i n i t i a l volume of solvent used f o r e x t r a c t i o n , and θ i s the volume of solvent r taken a f t e r c e n t r i f u g i n g as the a n a l y s i s a l i q u o t . Response f a c t o r s were determined by chromatographing a mixture of known weights of the t o x i c a n t s m y r i s t i c i n , f a l c a r i n o l , and f a l c a r i n d i o l , with a known weight of methyl palmitate (13) . Results and D i s c u s s i o n

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch021

t

Carrots (Daucus carota L.) make a s i g n i f i c a n t c o n t r i b u t i o n to the American d i e t by p r o v i d i n g f i b e r , minerals, and vitamins (14). These e s s e n t i a l n u t r i e n t s must be maintained at present l e v e l s or improved as the p l a n t breeder attempts to develop c a r r o t s of b e t t e r c u l i n a r y q u a l i t y . Other components of c a r r o t s , those that c o n t r i b u t e to the p l a n t ' s defense against damage, disease, and i n s e c t s , must be monitored to ensure that they do not increase or decrease to a troublesome l e v e l . Therefore, today more than ever, the p l a n t breeder and chemist need to cooperate i n developing new v a r i e t i e s . Most of the c a r r o t s grown i n the USA (80%) are f o r f r e s h market, the remainder are grown f o r processing ( 1 5 ) . An unestimated, but s i g n i f i c a n t , c a r r o t crop a l s o i s produced i n the home garden. The main t h r u s t of t h i s study involved four commercial v a r i e t i e s ( f o r f r e s h markets, Long Imperator 58 and High Color 9; f o r p r o c e s s i n g , Danvers 126 and Spartan Bonus) grown i n Wisconsin, F l o r i d a , C a l i f o r n i a , Arizona, and I l l i n o i s during 1979-1982. Other v a r i e t i e s commonly grown i n home gardens a l s o were examined (Royal Chantenay, Red Cored Chantenay, Gold Pak, Nantes H a l f Long) as w e l l as experimental genetic m a t e r i a l from the USDA Carrot Improvement Program (low v o l a t i l e inbreds B10138 and B9304, high v o l a t i l e inbreds B0493 and B3615, and white c a r r o t B10720). During the e a r l y stages of the study (1979-1980), only f a l c a r i n o l was measured; l a t e r measurements (1980-1982) included f a l c a r i n d i o l and m y r i s t i c i n . I d e n t i f i c a t i o n of peaks i n the chromatographic record was based on e l u t i o n times r e l a t i v e to methyl palmitate ( i n t e r n a l standard) i n nonpolar (3% SE-52) and intermediate p o l a r i t y (3% OV-17) packed columns. I d e n t i f i c a t i o n of m y r i s t i c i n , a small peak, was g e n e r a l l y confirmed by GC/MS.

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch021

338

XENOBIOTICS IN FOODS

A N D FEEDS

The data acquired were d i v i d e d i n t o three main groups, depending on sampling and a n a l y s i s p r o t o c o l , f o r f u r t h e r evaluation: (a) Type 1: 22 s e l e c t i o n s , with four samples each; sample weight was 50 g, with 4 c a r r o t s per sample. A l i q u o t s of each sample were analyzed. (b) Type 2: 5 s e l e c t i o n s , with 5 t o 18 c a r r o t s per sample. Carrots were quartered, and a l i q u o t s of two quarters each were analyzed i n duplicate. (c) Type 3: 28 s e l e c t i o n s with two samples each; sample weight was 300 t o 500 g, with 10 to 30 c a r r o t s per sample. A l i q u o t s of each sample were analyzed i n d u p l i c a t e . Although sampling procedures d i f f e r e d , there were 8 observations of each t o x i c a n t f o r each c a r r o t s e l e c t i o n . A n a l y s i s of variance was computed f o r each group; the l e a s t s i g n i f i c a n t d i f f e r e n c e (LSD, 0.05 l e v e l ) i s reported where appropriate (16) . The l a r g e s t sources of v a r i a t i o n i n the a n a l y s i s are due to sampling and l a c k of agreement between GC columns. Larger composite samples (Type 3) gave l e s s v a r i a t i o n f o r f a l c a r i n d i o l ; f a l c a r i n o l g e n e r a l l y gave good agreement with a l l sampling types. Agreement between GC columns a l s o was much b e t t e r f o r f a l c a r i n o l than f o r f a l c a r i n d i o l . One probable explanation f o r these r e s u l t s i s that the e l u t i o n time f o r f a l c a r i n o l i s very close to that of methyl p a l m i t a t e , whereas e l u t i o n time f o r f a l c a r i n d i o l was somewhat l a t e r . A l s o , i n the OV-17 column, establishment of a base l i n e a t the f a l c a r i n d i o l peak was complicated because of minor peaks that were not completely resolved from f a l c a r i n d i o l . Minor c o n s t i t u e n t s may have coeluted with f a l c a r i n d i o l . M y r i s t i c i n was detected i n only one v a r i e t y of c a r r o t s (Spartan Bonus) harvested i n Wisconsin i n 1981; the mean of 12 observations, 2 samples, was 1.4 mg/kg with a range of 1.3 to 1.5 mg/kg. Wulf e t a l . (9) a l s o report m y r i s t i c i n i n c a r r o t s . They show 15 mg/kg f o r the v a r i e t y Imperator, with l e s s e r amounts f o r other v a r i e t i e s . The p o l y a c e t y l e n e s , on the other hand, were found i n a l l v a r i e t i e s (Table I ) . The o v e r a l l mean of f a l c a r i n o l f o r 510 observations of a l l commercial v a r i e t i e s was 24.1 mg/kg; t h a t of f a l c a r i n d i o l f o r 389 observations was 65.1 mg/kg. Previous reports on t o x i c a n t l e v e l s , except f o r those r e l a t i n g to disease response, tended to overlook the f a l c a r i n d i o l content. The mean of the f a l c a r i n d i o l / f a l c a r i n o l r a t i o was 4.4, r e f l e c t i n g the higher concentration of f a l c a r i n d i o l . The standard e r r o r of a mean based on 2 samples (4 c a r r o t s per sample), 2 a l i q u o t s per sample, and 2 runs per sample, or 8 observations, was 2.8 f o r f a l c a r i n o l and 4.8 f o r f a l c a r i n d i o l . For s i n g l e observations, the r e s p e c t i v e standard d e v i a t i o n s were 15.2 and 24.8. About 70% of the v a r i a t i o n f o r f a l c a r i n o l was a s s o c i a t e d with sample, but only about 25% f o r f a l c a r i n d i o l . For f a l c a r i n d i o l , v a r i a t i o n a s s o c i a t e d with the assay d i f f e r e n c e s c o n t r i b u t e d most of the v a r i a t i o n . P r e c i s i o n f o r both t o x i c a n t s

YATES

21.

Table

ET AL.

I . Çummary

Analysis

of Carrot

o f Means o f F a l c a r i n o l

339

Constituents

and F a l c a r i n d i o l

for Thirteen

Carrot

Varieties-

Mean Falcarinol

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch021

Variety

mg

p e r kg

Falcarindiol mg

Ν

90

59.3 74.4

2.7 4.2

339

64.7

4.4

106.9 59.9 67.8

8.0

53.3 67.4

3.1 2.6 3.8

65.1

4.4

11.3 28. 1 28.2

85 92

28.1

Mean

460

24.3

Pak

Overall

3 mean-

Low v o l a t i l e s Low v o l a t i l e s

B10138 B9304

H i g h v o l a t i l e s B0493 H i g h v o l a t i l e s B3615 White c a r r o t B10720

13.4 22.2 22.6 25.6 22.1

10 8 8 24 50

510

24.1

389

12 8 44

Mean 3 O v e r a l l meansMinimum s i n g l e v a l u e Maximum s i n g l e v a l u e

-

10 8 8 24 50

8 8 8

564

10.5 12.3 6.1 9.2 9.4 9.5 22.7 0.4

falcarinol

9.0 2.8

104 114 124 118

Gold

falcarindiol/

54.5 69.0

72

High Color 9 Long I m p e r a t o r 58 D a n v e r s 126 S p a r t a n Bonus

N a n t e s H a l f Long Red C o r e d C h a n t e n a y Royal Chantenay Mean

p e r kg

ratio

8 8

78.0 38.0

8 12 8 44

58.9 284.6

433

76.8

53.5

129.1 132.9

16.7 384.2

2.7

7.6 3.1 9.8 52.4 14.2 20.6 7.1 0.8 82.9

H i g h C o l o r 9, L o n g I m p e r a t o r 58, D a n v e r s 126, and S p a r t a n Bonus v a r i e t i e s were grown i n W i s c o n s i n ( 1 9 7 9 - 1 9 8 2 ) , F l o r i d a ( 1 9 8 0 - 1 9 8 2 ) , C a l i f o r n i a (19801 9 8 2 ) , A r i z o n a ( 1 9 8 1 ) , and I l l i n o i s ( 1 9 8 1 - 1 9 8 2 ) . G o l d Pak, N a n t e s H a l f L o n g , Red C o r e d C h a n t e n a y , and R o y a l C h a n t e n a y v a r i e t i e s were grown i n I l l i n o i s ( 1 9 8 0 ) , and t h e e x p e r i m e n t a l g e n e t i c m a t e r i a l s were grown i n W i s c o n s i n (19801981).

2 - Ν Number o f a n a l y s e s

ignoring

3 - Means o f a l l v a r i e t i e s

listed

sampling design. above.

XENOBIOTICS IN FOODS A N D F E E D S

Table I I . F a l c a r i n o l and F a l c a r i n d i o l Means by L o c a t i o n , Year, and V a r i e t y

Falcarinol

Falcarindiol

mg/kg

mg/kg

15.61 15.1 33.5 25.2 5.0

75.1 52.4 62.8 56.2 8.8

22.5 25.9 25.4 5.0

65.8 57.6 6.2

12.1 28.8 27.9 29.6 5.8

46.9 65.2 57.4 70.2 8.8

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch021

Location I l l i n o i s (NRRC) Wisconsin Florida California LSD-

Year 1980 1981 1982 LSD

Variety High Color 9 Long Imperator 58 Danvers 126 Spartan Bonus LSD

- Based on 1981 and 1982 only. 2 d i f f e r e n c e (0.05 l e v e l ) - Least s i g n i f i c a n t between two means

21.

YATES ET AL.

Analysis

of Carrot

Table I I I . F a l c a r i n o l

341

Constituents

and F a l c a r i n d i o l

Means A s s o c i a t e d with the I n t e r a c t i o n of L o c a t i o n and Year^

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch021

Year Location

1980

1981

1982

F a l c a r i n o l mg/kg Wisconsin Florida California LSD-= 8.6

18.0 13.4 13.8

27.2 40.5 32.8

21.9 23.7 29.6

F a l c a r i n d i o l mg/kg Illinois Wisconsin Florida California LSD =12.4

95.3 56.8 56.9 54.0

54.9 48.1 68.8 58.4

- Each mean i s f o r High Color 9, Long Imperator 58, Danvers 126, and Spartan Bonus v a r i e t i e s . 2 - Least s i g n i f i c a n t d i f f e r e n c e (0.05 l e v e l ) between two means.

- 1981, 1982.

- 1980, 1981, 1982.

69.6 67.6 90.0 59.2

49.6 60.4 81.6 48.8

High Color 9 Long Imperator 58 Danvers 126 Spartan Bonus LSD =16.8

Florida

56.8 71.2 74.8 68.4

F a l c a r i n d i o l mg/kg-

14.9 37.1 40.5 41.5

F a l c a r i n o l mg/kg-

Wisconsin

6.2 22.3 18.6 13.3

Illinois

High Color 9 Long Imperator 58 Danvers 126 Spartan Bonus LSD =10.0

Variety

48.8 51.6 54.0 33.2

13.2 29.8 22.5 34.8

California

Table IV. F a l c a r i n o l and F a l c a r i n d i o l Means by V a r i e t y and L o c a t i o n

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch021

Ζ D τι m m α

>

σ

Ο Ο

Τ]

π

S

00

x m Ζ ο

Ν)

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch021

21.

YATES E T A L .

Analysis

of Carrot

Constituents

343

was improved by a f a c t o r of 2 when sample s i z e was increased from 4 to 18 c a r r o t s . The means by l o c a t i o n , year, and v a r i e t y f o r four s e l e c t e d commercial v a r i e t i e s are shown i n Table I I . The combination of data f o r a l l years and v a r i e t i e s shows that the F l o r i d a l o c a t i o n produced the highest c o n c e n t r a t i o n o f f a l c a r i n o l . However, l o c a t i o n d i f f e r e n c e s are dependent on year; the high value of f a l c a r i n o l occurred a t a d i f f e r e n t l o c a t i o n each year (Table I I I ) . Table I I a l s o shows that f a l c a r i n d i o l c o n c e n t r a t i o n i s highest i n those c a r r o t s grown i n I l l i n o i s ; however, Table I I I again shows that the high t o x i c a n t l e v e l i s i n a d i f f e r e n t l o c a t i o n each year. Therefore, the s i g n i f i c a n t e f f e c t s of l o c a t i o n i n Table I I must be judged i n the l i g h t of a s i g n i f i c a n t year-location interaction. Tables II and I I I i n d i c a t e that the l e v e l s of both t o x i c a n t s were highest i n 1981. Perhaps these r e s u l t s account f o r v a r i a t i o n i n r e s i s t a n c e to r o t that i s observed i n c a r r o t s from year to year ( 6). The i n t e r a c t i o n o f v a r i e t i e s with year was not s i g n i f i c a n t , suggesting that v a r i e t a l d i f f e r e n c e s tended to remain the same from year to year. An i n t e r a c t i o n of v a r i e t y and l o c a t i o n was observed f o r f a l c a r i n o l (Table IV). Long Imperator 58, Danvers 126, and Spartan Bonus show considerable v a r i a t i o n among l o c a t i o n s , but High Color 9 has the lowest values a t a l l three l o c a t i o n s . High Color 9 has low l e v e l s of f a l c a r i n d i o l , whereas Danvers 126 c o n s i s t e n t l y had high l e v e l s . Sensory scores on a scale of 1 t o 3 were t a b u l a t e d by one of the authors with a l i m i t e d number o f c a r r o t s f o r four sensory evaluations: sweetness, harsh a f t e r t a s t e , c r i s p n e s s , and o v e r a l l preference. There was no i n d i c a t i o n o f a s s o c i a t i o n between t o x i c a n t l e v e l s and sensory e v a l u a t i o n s i n a p l o t of t o x i c a n t s versus sensory e v a l u a t i o n . Therefore, i t seems h i g h l y probable that c u l i n a r y q u a l i t y can be improved without increasing toxicant levels.

Literature Cited 1. Crosby, D. G.; Aharonson, N. Tetrahedron 1967, 23, 465-472. 2. Bentley, R. K.; Thaller, V. Chem. Commun. 1967, 439-440. 3. Bohlmann, F.; Niedballa, V.; Rode, Κ. M. Chem. Ber. 1966, 99, 3552-3558. 4. Bentley, R. K.; Bahattacharjee, D.; Jones, E. R. H.; Thaller, V. J. Chem. Soc. C 1969, 685-688. 5. Garrod, B.; Lewis, B. G. Trans. Br. Mycol. Soc. 1980, 75, 166-169. 6. Harding, V. K.; Heale, J. B. Ann. Appl. Biol. 1981, 99, 375-383. 7. Garrod, B.; Lewis, B. G.; Brittain, M. J.; Davies, W. P. New Phytol. 1982, 90, 99-108.

344

8. 9. 10. 11. 12. 13.

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch021

14. 15.

16.

X E N O B I O T I C S IN F O O D S A N D F E E D S

Ho, A. K. S. Dept. Basic Sciences, University of Illinois College of Medicine, Peoria School of Medicine, Peoria, Illinois 61656, unpublished data, 1982. Wulf, L. W.; Nagel, C. W.; Branen, A. L. J. Agric. Food Chem. 1978, 26, 1390-1393. Forrest, J. E.; Heacock, R. A. Lloydia 1972, 35, 440-449. Fuhremann, T. W.; Lichtenstein, E. P. J. Agric. Food Chem. 1979, 27, 87-91. Surak, J. G. Proc. Fla. State Hort. Soc. 1978, 91, 256-258. Yates, S. G.; England, R. E. J. Agric. Food Chem. 1982, 30, 317-320. Senti, F. R.; Rizek, R. L. Crop Soc. Am. Spec. Publ., No. 5, 1974, 7-20. Simon, P. W.; Peterson, C. E.; Lindsay, R. C. "Quality of Selected Fruits and Vegetables of North America"; Teranishi, R.; Barrera-Benitez, Η., Eds.; ACS SYMPOSIUM SERIES, No. 170, ACS: Washington, D.C., 1981; p. 109-118. Steel, R. G. D.; Torrie, J. H. "Principles and Procedures of Statistics," 2nd ed., McGraw-Hill Book Co., NY, 1980.

RECEIVED July

6, 1983

22 Ingestion of Pyrrolizidine Alkaloids: A Health Hazard of Global Proportions JAMES N. ROITMAN

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch022

Natural Products Chemistry Research, Western Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, Berkeley, CA 94710 Pyrrolizidine alkaloids, about 200 of which have been identified, occur in a number of unrelated plant families distributed throughout the world. These alkaloids have been shown to be responsible for the long known hepatotoxicity in humans and animals associated with ingestion of certain plants. Because the onset of symptoms often occurs only after considerable time has elapsed (up to several years), it is likely that many, if not most, cases of pyrrolizidine alkaloid-caused liver damage are ascribed to other causes. The diverse physiological effects as well as the underlying mode of toxicity are presented. The chemical properties and analytical methods employed are summarized and the manner in which pyrrolizidine alkaloids enter the human food chain via contaminated foodstuff and the use of herbs is discussed as well as the nature of the problem in the U.S. today. Reasons for increasing consumption of herbal teas are suggested. I t was eighty years ago that G i l r u t h demonstrated by feeding experiments that tansy ragwort (Senecio jacobaea) was r e s p o n s i b l e f o r a disease o f horses and c a t t l e i n New Zealand c a l l e d Winton d i sease ( 1 ) . Other feeding experiments e s t a b l i s h e d that c e r t a i n species o f Senecio (Compositae) were r e s p o n s i b l e f o r P i c t o u d i sease of c a t t l e (Canada, 1906) ( 2 ) , Molteno disease (Cape Colony, South A f r i c a , 1904) ( 3 ) , "dunsiekte" disease of horses (South A f r i c a , 1918) ( 4 ) , and "walking d i s e a s e " of horses (northwestern Nebraska, 1929) ( 5 ) . Feeding experiments with various species of C r o t a l a r i a (Leguminosae) established the e t i o l o g y of s e v e r a l diseases of horses e x h i b i t i n g symptoms s i m i l a r to those caused by Senecio although the genera are u n r e l a t e d b o t a n i c a l l y . Diseases known as " j a a g s i e k t e " i n S. A f r i c a and "walkabout" i n A u s t r a l i a This chapter not subject to U . S . copyright. P u b l i s h e d 1983, A m e r i c a n C h e m i c a l S o c i e t y

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch022

346

XENOBIOTICS IN FOODS A N D F E E D S

were reproduced by feeding horses C r o t a l a r i a dura and C. retusa r e s p e c t i v e l y 06,^). A number of p l a n t s i n the f a m i l y Boraginaceae were a l s o demonstrated to cause assorted r e l a t e d diseases: H e l i o tropium species were r e s p o n s i b l e f o r l i v e r dystrophy prevalent i n c e n t r a l A s i a during the period 1931-45 i n man and domestic animals (8-12) as w e l l as sheep poisoning i n A u s t r a l i a (13); Trichodesma incanum was r e s p o n s i b l e f o r an A s i a n disease of horses and c a t t l e , c a l l e d " s u i l j u k " (14), Echium l y c o p s i s caused sheep poisoning i n A u s t r a l i a (13,15), and Amsinckia intermedia produced c i r r h o s i s i n horses and "hard l i v e r d i s e a s e " i n c a t t l e and pigs i n the P a c i f i c Northwest of the U.S. (16). Although the p l a n t s a s s o c i a t e d with these v a r i o u s , o f t e n l e t h a l , diseases occur i n three unrelated plant f a m i l i e s , a common feature of a l l i s t h e i r production of p y r r o l i z i d i n e a l k a l o i d s . These have been shown to be capable of causing the p h y s i o l o g i c a l changes c h a r a c t e r i s t i c of the abovementioned diseases although other p l a n t c o n s t i t u e n t s may play a r o l e i n exacerbating or mediating the e f f e c t s of the a l k a l o i d s and may i n part e x p l a i n some of the i n c o n s i s t e n c i e s observed i n animal feeding experiments. Chemistry of P y r r o l i z i d i n e A l k a l o i d s There are c u r r e n t l y about 200 p y r r o l i z i d i n e a l k a l o i d s known, most of which are mono- and d i - e s t e r s of the saturated and 1,2unsaturated amino-aleohoIs (commonly known as necines or necine bases) shown i n Figure 1 (17a). Numerous t o x i c o l o g i c a l s t u d i e s on small l a b o r a t o r y animals have shown that acute and chronic t o x i c i t y are caused only by e s t e r i f i e d 1,2-unsaturated necines although an unrelated per-acute syndrome, manifested by r a p i d m o r t a l i t y , can be induced by very l a r g e doses of saturated necine e s t e r s (17b,18). The s t r u c t u r e s of some r e p r e s e n t a t i v e p y r r o l i z i d i n e a l k a l o i d s are shown i n Figures 2 thru 4. The e s t e r i f y i n g a c i d s are h i g h l y branched and i n f r e q u e n t l y found elsewhere i n nature. Although the s t r u c t u r e s are not e s p e c i a l l y complex, the number of asymmetric centers renders unequivocal s t r u c t u r e d e t e r mination d i f f i c u l t . Furthermore, mixtures of diastereomers and geometric isomers o f t e n c o e x i s t i n p l a n t e x t r a c t s , and separation of these i s laborious and involves s p e c i a l techniques. These problems confounded a number of the e a r l y chemical i n v e s t i g a t i o n s e s p e c i a l l y s i n c e mixtures of the a l k a l o i d s o f t e n c r y s t a l l i z e as i f they were s i n g l e substances. An a d d i t i o n a l feature of p y r r o l i z i d i n e a l k a l o i d s i s that they o f t e n occur admixed with t h e i r N-oxides. The l a t t e r are h i g h l y water s o l u b l e and consequently are not extracted from aqueous s o l u t i o n s by the normal e x t r a c t i o n procedures employed to i s o l a t e a l k a l o i d s . However t o t a l p l a n t a l k a l o i d s may be 90% i n the form of N-oxides, rendering meaningless any estimates of a l k a l o i d content determined by normal e x t r a c t i o n techniques. Chemical reducing agents added during i s o l a t i o n procedures r a p i d l y convert Noxides to the corresponding a l k a l o i d bases allowing i s o l a t i o n from aqueous a l k a l i n e s o l u t i o n by solvent e x t r a c t i o n (17c).

ROiTMAN

HO

u

Ingestion

of Pyrrolizidine

Alkaloids

CHjOH

CH OH 2

CD

CD HELIOTRIDINE

Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch022

RETRONECINE

HO

CH OH 2

H

SUPINIDINE

CH OH

H

2

°

y

^ 2°H H

Ct> CD

CH,

LINDELOFIDINE (3 OTHER STEREOISOMERS

OTONECINE

PLATYNECINE

ALSO OCCUR NATURALLY)

HO

y

CH OH 2

ct> DIHYDROXY HELIOTRIDANE

HO

H

CH OH 2

ct> ROSMARINECINE

Figure 1. Structures of necine bases, esters of which are hepatotoxic (retrone cine, heliotridine, supinidine, otonecine) or nonhepatotoxic (platynecine, dihy droxyheliotridane, rosmarinecine, lindelofidine) pyrrolizidine alkaloids.

American Chemical Society Library 1155

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XENOBIOTICS IN F O O D S A N D F E E D S

19

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C = C - C H - C —C«'..CH "CO C H 16ÇO !7Q CH 0 2

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Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch022

C= C-CH -C—C""CH / CH ι I ÇH 0 ο

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Publication Date: October 25, 1983 | doi: 10.1021/bk-1983-0234.ch023

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