Handbook of Food Toxicology (Food Science and Technology)

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ISBN: 0-8247-0760-5 This book is printed on acid-free paper. Headquarters Marcel Dekker, Inc. 270 Madison Avenue, New York, NY 10016 tel: 212-696-9000; fax: 212-685-4540 Eastern Hemisphere Distribution Marcel Dekker AG Hutgasse 4, Postfach 812, CH-4001 Basel, Switzerland tel: 41-61-260-6300; fax: 41-61-260-6333 World Wide Web http://www.dekker.com The publisher offers discounts on this book when ordered in bulk quantities. For more information, write to Special Sales/Professional Marketing at the headquarters address above. Copyright © 2002 by Marcel Dekker, Inc. All Rights Reserved. Neither this book nor any part may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, microfilming, and recording, or by any information storage and retrieval system, without permission in writing from the publisher. Current printing (last digit): 10 9 8 7 6 5 4 3 2 1 PRINTED IN THE UNITED STATES OF AMERICA

Copyright 2002 by Marcel Dekker. All Rights Reserved.

To all those who appreciate my sole existence, especially my wife Usha and daughter Maithili and To Ratan, Shastry, Suresh, and Prakash, for being such wonderful friends through thick and thin


Preface

Food toxicology is deeply rooted in the history of civilization. In their quest for food, our ancestors must have attempted to eat a variety of plants and animals and soon recognized that there were harmful as well as beneficial effects of their consumption. The selection by nomadic humans of only a handful of plant species for domestication and cultivation from the estimated 350,000 species of plants documented in the annals of botany and plant sciences is certainly not a chance occurrence. Gathering peoples perhaps accidentally found the very species that were the most predisposed to domestication, as well as the most well-suited to fulfill human nutritional requirements. Our experience throughout history has taught us much about how to avoid injury from consuming natural products. We now know which products not to eat under any circumstances, which can be eaten under some circumstances, and how to process other products to render them safe for consumption. History has thus taught us how to classify all substances into two classes: those that are safe and those that are harmful or poisonous. Such a classification, however, is not warranted in a strict scientific sense, primarily because a strict line of demarcation classifying and separating beneficial and harmful chemicals cannot be drawn, and because the degree of harmfulness of any compound is essentially related to the amount consumed. In fact, over 400 years ago Paracelsus pointed out that “all substances are poisons; there is none which is not a poison. The right does differentiates a poison and a remedy.” Indeed, the entire concept of toxicity needs to be evaluated from the viewpoint of a risk/benefit concept associated with the


consumption of any given material. There is no such thing as absolute safety. Our daily lives are still shaped by our acceptance of numerous acceptable risks. Nearly everything we consume, including salt, sugar, starch, fat, protein, some minerals and vitamins, and even water, has a harmful effect when consumed in high enough concentration. Hazardous substances associated with food include toxic or antinutritive compounds that are naturally present in plants and animals; toxins that are produced ruing processing; incidental contaminants such as pesticides, antibiotic drug residues, and environmental pollutants; and foodborne pathogens. However, this does not mean that food is hazardous to human beings. Toxic components in foods—although they should indeed be minimized—are inevitable hazards of living. A substance that is considered to be toxic/antinutritive has a more or less pronounced capacity to induce deleterious effects on an organism when tested by itself in certain doses. This does not always happen under usual dietary conditions. We consume many toxic substances in our normal diet every day without showing any signs of intoxication. This is probably because natural toxicants usually exert their effects only when other potentiating substances are available. Also, the concentration of these compounds occurring naturally in food is often so low that the item must be consumed in extraordinarily large amounts daily over a prolonged period for intoxication to occur. Similarly, most toxic effects of potentially hazardous chemicals are not additive. In fact, antagonistic reactions that make some ingredients interfere and reduce the toxic

effects of other components are not unusual. Thus, many natural products that are common in the human diet have found wide acceptance not because they are free of toxic substances, but because they do not contain enough toxins to be harmful when consumed in reasonable quantities as part of a balanced diet—or because cooking or another process eliminates their toxic activity. In the vast majority of instances, our food supply is quite wholesome. In the unfortunate incidents when some link(s) in the food production, processing, and distribution scheme fail(s), such foods, when consumed, have produced adverse toxic responses that vary in severity from insignificant to fatal. As compared with naturally occurring toxic/antinutritive compounds in the human food chain, the situation is quite different with microbial contaminant of foods. In fact, perhaps the greatest damage, in terms of both mortality and morbidity worldwide, can be directly attributed to microbial contamination. Although changes and improvements in food processing operations as well as in sanitary practices have contributed to an important increase in the life span of humans in the last century, these significant improvements are now challenged by the appearance of microbes resistant to multiple antibiotics (e.g., Salmonella sp. and the emergence of new bacterial and fungal pathogens (e.g., Campylobacter, Listeria, E. Coli 0157:H7, fumonisins). In the United States alone, between 6.5 million and 81 million cases of foodborne illness and as many as 10,000 related deaths from seven major foodborne pathogens occur each year, costing $6.6 billion to $37.1 billion in economic losses. The situation is grim even in developing countries where water-borne and food-borne diseases such as cholera, jaundice, and diarrhea—which impair human health to a great degree, and therefore the body’s efficiency of food absorption—are perhaps more important factors affecting human health than many naturally occurring toxic/antinutritive compounds in the food chain. These effects are further magnified by a shortage of such basic commodities as a clean and safe supply of drinking water and adequate food for subsistence—this alone was good enough motivation for me to undertake this project. The primary aims for Handbook of Food Toxicology are twofold. (1) to provide basic coverage of the principles of toxicology relevant to food science and nutrition, and (2) to provide the latest information on various toxic and microbial hazards associated with modern-day foods. This book is divided into two parts that comprise a total of 18 chapters. The first part, consisting of Chapters 1–6, deals with the science and principles of toxicology, manifestations of toxic effects, biotransformations of toxicants relevant to food science and human nutrition, and some of the


regulatory and QA/QC issues. Chapters 7 through 18 describe the basic aspects of toxicity associated with commonly occurring dietary components and substances (naturally occurring, intentionally added, or incidental), as well as those associated with microbial contamination of foods. A basic understanding of the principles behind the occurrence of microbes in the food chain and their toxicity or toxic mechanisms not only allows us to appreciate the complexity of our food supply but is essential for developing newer and safer food production, processing, handling, and distribution technologies. No single food toxicology book can cover all aspects of the toxicity and safety of the myriad of food used in many different ways by humans worldwide. Indeed, volumes and monographs are available on the topic of practically every chapter in this book, and even on those of many of the chapter sections. Every effort, however, was made to cover important toxic hazards associated with food consumption. For some toxins, only historical viewpoints are described, since research during the past decade on many of these compounds (e.g., flavonoids, phytates, antioxidants) has shown several positive health benefits associated with their consumption as part of a normal, wellbalanced diet. In contrast, in-depth coverage is provided on microbial toxins and food pathogens, since these appear to be the predominant causes of morbidity and mortality associated with our food supply. Hopefully, this book represents a compromise between the historical views associated with the traditional, well-known toxic components found in our food supply and the exciting new developments occurring on several other fronts, especially on foodborne infections and intoxications. It is my sincere hope that the information presented in this book will serve professionals in many disciplines, including agriculture, food science, nutrition, microbiology, toxicology, public health, medicine, and other health-related areas. Selected chapters can also be used as college-level teaching material. Finally, it is inevitable in a book of this breadth that omissions, occasional errors, and lapses in the accuracy of interpretation will have escaped the detection of even the most assiduous proofreaders. I hope that any such mistakes are both minor and minimal, and I accept full and exclusive responsibility for them. I welcome comments and suggestions for improvement and for correction of any errors. Sincere appreciation is extended to the editorial and production staff of Marcel Dekker, Inc., especially to Ms. Maria Allegra, Ms. Lila Harris, Ms. Katie Stence, Ms. Theresa Stockton, and Ms. Susan Thornton. Without their cooperation and tremendous patience, this book would

never have been written. I gratefully acknowledge the original treatise in this field: the late Professor Jose M. Concon’s groundbreaking two-volume Food Toxicology, published in 1988. In fact, the origin of this book can be traced back to his monumental work in the field. I am also greatly indebted to Professor D. K. Salunkhe of Utah State University, who first encouraged me to undertake this task.


Finally, no words will ever fully describe the untiring and continued support and encouragement provided by my wife Usha and daughter Maithili during the arduous task of putting together this book.

S. S. Deshpande

Contents

Preface SCIENCE AND PRINCIPLES OF TOXICOLOGY 1.

The Science of Toxicology

2.

Principles of Toxicology

3.

Manifestations of Organ Toxicity

4.

Carcinogenesis, Mutagenesis, and Teratogenesis

5.

Biotransformation of Xenobiotics

6.

Measurement of Toxicants and Toxicity TOXICITY IN FOODS

7.

Dietary Constituents

8.

Food Additives

9.

Toxicants Resulting from Food Processing

10.

Toxicants and Antinutrients in Plant Foods

11.

Fungal Toxins

12.

Food-Borne Infections


13.

Bacterial Toxins

14.

Seafood Toxins and Poisoning

15.

Mushroom Toxins

16.

Toxic Metals, Radionuclides, and Food Packaging Contaminants

17.

Pesticides and Industrial Contaminants

18.

Drug Residues


1 The Science of Toxicology

1.1

INTRODUCTION

The origins of toxicology appear to be deeply rooted in the history of human civilization. Our ancestors in their quest for food must have attempted to eat a variety of foods of both plant and animal origin and soon recognized that there were harmful as well as beneficial consequences associated with the consumption of such material. The rise of agricultural knowledge has been traced back to ancient times, when humankind made the transition from nomadic hunting/gathering tribes to more settled societies supported by domesticated animal herds and cultivated crops. In terms of archaeological findings, primitive agriculture may have developed as early as 9000–7000 B.C. in the Near East (Garfield, 1990). The selection of only a handful of plant species for domestication and cultivation by the nomadic human is certainly not a chance occurrence. It must predate agriculture by at least some thousands of years. Its enormous complexity is further illustrated by the 3000-plus species of the estimated 350,000 species of plants documented in the annals of botany and plant sciences that have been used historically in some form to feed humans (Deshpande, 1992; Borlaug, 1981; Wittwer, 1980). Fewer than 300 are used currently worldwide in organized agriculture. Among these, at least 150 different species are grown in sufficient quantities to enter the world trade. In contrast, Wittwer (1980) suggests, today some 24 crops essentially stand between people and starvation. In approximate order of importance these crops are rice, wheat, corn, potato, barley, sweet potato, cassava, soybean, oat, sorghum, millet, sugarcane, sugar beet, rye, peanut, field bean, chick-


pea, pigeon pea, mung bean, cowpea, broad bean, yam, banana, and coconut. Although cereals with nine species and legumes with eight constitute the preponderance of the global food production, together they constitute only 0.005% of the available wealth in the plant kingdom. Present-day humans, no doubt, have diversified the uses of important economic plants greatly. They, however, have added relatively little to this list of basic staples. Domestication of only such a few plant species as human food sources is truly one of the most extraordinary stories of our history. Beginning only by collecting, the gathering peoples perhaps accidentally(?) chanced upon the very species that were the most predisposed to domestication and were well suited to fulfill human nutritional requirements. Our experience throughout history has thus taught us much about how to prevent injury from consuming natural products as foods. We now know which products not to eat under any circumstances, which can be eaten under some circumstances, and how to process certain other products to render them safe for consumption. History has thus taught us how to classify all substances in two classes: those that are safe and the others that are harmful. Traditionally, the term food was used for those materials that were beneficial and essential for the functioning of human body. Substances that were distinctly harmful to the body were classified as poisons. This concept involving the division of chemicals into two categories has persisted to the present day. It readily places certain biological and botanical and, in fact, all distinctly harmful chemicals into a category that is accorded due respect. Loomis (1978), however, suggested that such a classification, in a strictly scientific sense, is

not warranted, primarily because a strict line of demarcation classifying and separating the beneficial and harmful chemicals cannot be drawn and because the degree of harmfulness of any compound is essentially related to the amount consumed. Indeed, the entire concept of toxicity needs to be evaluated from the viewpoint of a risk/benefit concept associated with the consumption of any given material. In fact, Paracelsus (1493–1541) over 400 years ago pointed out that “all substances are poisons; there is none which is not a poison. The right dose differentiates a poison and a remedy.” Since all substances can produce injury or death under some exposure conditions, it is evident that there is no such thing as an absolute safe substance or chemical that will be free of injurious effects under all conditions of exposure. As a corollary, it is also true that there is no chemical that cannot be used safely by limiting the dose or exposure. Our daily lives are still shaped by our acceptance of numerous acceptable risks. Nearly everything we consume, including salt, sugar, starch, fat, protein, some minerals and vitamins, and even water, has a harmful effect when consumed in high enough concentration. However, this does not necessarily mean that the substance is hazardous to human beings. Toxic compounds in our foods, medicines, and environment—though they should indeed be minimized—are inevitable hazards of living. A substance that is considered to be toxic/antinutritive has a more or less pronounced capacity to induce deleterious effects on the organism when tested by itself in certain doses. This does not always happen under the usual dietary conditions. We consume many toxic substances in our normal diet every day without showing any signs of intoxification. This is probably because natural toxicants usually exert their effects only when they are consumed under special conditions or when there are other potentiating substances present. Also, the concentration of these compounds occurring naturally in the food is often so low that the item must be consumed in usually unrealistically large amounts every day for a prolonged period for intoxification to occur. Furthermore, humans can handle small amounts of various toxicants. Similarly, most toxic effects of various chemicals that are potentially hazardous do not have an additive effect. In fact, antagonistic reactions that make some ingredients interfere with and reduce the toxic effects of other components are not unusual. Thus, many natural products that are common articles of the diet have found wide acceptance, not because they are free of toxic substances, but because they do not contain enough to be harmful when consumed in reasonable quantities as a part of a balanced diet, or because cooking or some other process eliminates their toxic activity.


It is thus the “dose” of any given substance that determines its degree of harmfulness. If a sufficient dose is ingested or is in contact with a biological mechanism, then a harmful or toxic effect will be the consequence in the sense that the ability of that biological mechanism to carry on a function is either seriously impaired or destroyed. Such harmful effects often do not occur suddenly as the dose is increased from minimal to maximal levels. Rather a graded response related to progressive changes in dose is observed. The basic premise of the science of toxicology with respect to any biological effects of a chemical agent is to study this relationship that exists between the dose or concentration and the response that is obtained. Furthermore, toxic chemicals may be selective or nonselective in exerting their harmful effects on biological systems. Thus selective chemicals may exert their harmful effects only in a few living species, primarily because some protective mechanisms may be present in the resistant species. In contrast, those that act nonselectively exert an undesirable or harmful effect on all living matter. Fortunately, such compounds seldom find their way into our food chain under normal conditions. The term toxicology is derived from Latin and Greek origins (Latin toxicum meaning “poison”; Greek toxikom meaning “arrow poison”; and Latin logia, meaning “science” or “study”) and literally means a study of poisons in relation to living organisms. The science of toxicology therefore can be best approached as the study of the effects of chemicals on biological systems, with emphasis on the mechanisms of harmful effects of chemicals and the conditions under which such harmful effects can occur. In its broadest sense, it also includes socioeconomic considerations as well as legal ramifications.

1.2

HISTORY OF TOXICOLOGY

Ironically, toxicology must rank as one of the oldest practical sciences because humans, from the very beginning, needed to avoid the numerous toxic plants and animals in their environment. The Egyptian Ebers Papyrus (circa 1500 B.C.) and the Hindu Vedas (circa 5000 B.C.) rank as two of the earliest surviving medical records that contain information on several poisons. The surviving medical works of Hippocrates, Aristotle, and Theophrastus, published during the period 400–250 B.C., all included some mention of poisons. Dioscorides, a Greek employed by the Roman emperor Nero in about A.D. 50, first attempted to classify plants according to their toxic and therapeutic effects. There appear to have been very few noteworthy advances in either medicine or toxicology during the Middle

Ages. The only significant contribution to toxicology during this period appears to be that of Moses ben Maimon, or Maimonides (1135–1204). He published a treatise, Poisons and Their Antidotes, a first-aid guide to the treatment of accidental or intentional poisonings and insect, snake, and mad dog bites. Maimonides recommended that suction can be applied to insect stings or animal bites as a means of extracting the poison and advised application of a tight bandage above a wound located on a limb. He also noted that the absorption of toxins from the stomach could be delayed by ingestion of oily substances such as milk, butter, or cream. On the basis of critical and cautious observations, he also rejected numerous popular remedies of the day after finding them to be ineffective and mentioned his doubts concerning the efficacy of others. During the later Middle Ages, Philippus Aureolus Theophrastus Bombastus von Hohenheim Paracelsus (1493–1541) made revolutionary contributions to this discipline we now call toxicology. He first proposed the toxic agent as a chemical entity and was clearly aware of the dose-response relationship. The following four principles he first laid out still form the core of toxicological sciences. 1. 2. 3. 4.

Experimentation is essential in examination of responses to chemicals. A distinction must be made between therapeutic and toxic properties of chemicals. These properties are sometimes, but not always, indistinguishable except by dose. One can ascertain a degree of specificity of chemicals and their therapeutic or toxic effects.

Mathieu Joseph Bonaventura Orfila (1787–1853), a Spanish physician to Louis XVIII of France and a teacher at the University of Paris, is often cited as the father of modern toxicology. Orfila was the first to attempt a systematic correlation between the chemical and biological information of the then well-known poisons. He also singled out toxicology as a discipline distinct from others and defined it as the study of poisons. He wrote in 1815 the first book of general toxicology that was exclusively devoted to adverse effects of chemicals. Concerned with legal implications of poisoning, Orfila also pointed out the importance of determining a chemical analysis to establish a definitive cause of poisoning. Some of the analytical procedures he developed are still in use today. Since Orfila’s pioneering work, developments in toxicology slowly evolved. Although toxicology’s origins predate those of most other biological sciences and perhaps even those of medicine, most of the useful information related to modern toxicology has only been learned since the turn of the 20th century. In fact, the emergence of


toxicology as a distinct discipline is a much more recent phenomenon. There are many reasons for this, including the development of new analytical methods since the end of the Second World War, the emphasis on drug testing that followed the thalidomide tragedy, the focus on pesticide testing since the publication of Rachel Carson’s Silent Spring, public concerns about hazardous waste disposal, and, more recently, the increased incidence of food poisonings by microbial pathogens. Detailed descriptions of the historical developments in the field of toxicology can be found in several excellent reviews (Loomis, 1978; Holmstedt and Liljestrand, 1981; Doull and Bruce, 1986).

1.3

SCOPE/DIVERSITY OF TOXICOLOGY

Modern toxicology is a multidisciplinary field, which has extracted many of the principles and techniques from several basic biological and chemical sciences (Figure 1.1). It is primarily an applied science, dedicated to the enhancement of the quality of life and the protection of the environment. It draws heavily on tools of chemistry and biochemistry. Those of chemistry provide analytical methods for toxic compounds, particularly for forensic toxicology and residue analysis, and those of biochemistry provide the techniques to investigate the metabolism and mode of action of toxic compounds. Toxicology may also be considered an area of fundamental biology since the adaptation of organisms to toxic environments has important implications for ecology and evolution. Sciences such as immunology, biomathematics, and ecology are also important, but to a more limited extent. The science of toxicology contributes heavily in the field of medicine, especially forensic medicine, clinical toxicology, pharmacy and pharmacology, public health, and industrial hygiene. It also contributes in an important way to veterinary medicine and to such aspects of agriculture as the development and safe use of agrochemicals. Its contributions to the field of environmental studies are among the most rapidly expanding areas in the world today. Thus, any attempt to define the scope of toxicology must take into account the fact that the various subdisciplines shown in Figure 1.1 are not mutually exclusive and frequently are heavily interdependent. Because of the overlapping of mechanisms, chemical classes, and use classes and effects, clear division into distinct branches of toxicology is often not possible. However, for the sake of convenience, subdivisions of toxicology can be defined by following any of the several classifications suggested by

Figure 1.1

The relationship of toxicology to basic sciences, its evolution and applications.

Hodgson (1987). These are briefly described in the following sections. 1.3.1 Applied Toxicology Based on Disciplines Various aspects of applied toxicology can be defined as they occur in or relate to a particular field (Figure 1.1). These include the following.

Veterinary Toxicology Veterinary toxicology is to animals what clinical toxicology is to humans. It deals with the diagnosis and treatment of the poisoning of animals, particularly livestock and household pets. An important concern of veterinary toxicology is the possible transmission of toxins and pathogens to the human population via meat, fish, milk, and other foodstuffs. Environmental Toxicology

Clinical and Forensic Toxicology Clinical toxicology is concerned with the diagnosis and treatment of human poisoning. It encompasses the study of chemicals originating from any and all sources and is primarily concerned with all aspects of the interaction between these chemicals and people. Forensic toxicology, in contrast, combines analytical chemistry with essential toxicological principles in order to deal with the medicolegal aspects of the toxic effects of drugs and chemicals on humans. Its primary role is to aid in establishing cause-effect relationships between exposure to a drug or poison and the toxic or lethal effects of the compound. It relies heavily on specific and highly sensitive analytical methods, which can efficiently isolate, identify, and quantitate the toxic compound in question from clinical and other samples.


The broad discipline of environmental toxicology encompasses the study of chemicals that are the contaminants of food, water, soil, or the atmosphere. It is primarily concerned with the movement of chemicals and toxicants and their metabolites in the environment and in food chain, and the effect of such toxicants on individuals and populations. Environmental toxicology is also concerned with toxic substances that may enter the lakes, streams, rivers, and oceans. The most common problems dealt with in this aspect of toxicology are water-borne viruses and bacteria, radioactive waste, sewage eutrophication, and industrial pollutants. Occupational/Industrial Toxicology Occupational/ industrial toxicology is a specific area of environmental toxicology that has grown out of a need to

protect the worker from poisonous substances and, in general, to make the working environment safe. The objective obviously is to prevent impairment of health of an individual while on the job. In the United States, the Occupational Safety and Health Act (OSHA) was passed in 1970 to protect the health of workers. Two agencies, the National Institute for Occupational Safety and Health (NIOSH), responsible for developing safety and health standards, and the American Conference of Governmental Industrial Hygienists (ACGIH), devoted to setting safety standards for chemicals in the working environment, are primarily responsible for the enactment of OSHA guidelines under the jurisdiction of the Department of Labor. 1.3.2 Classification Based on Measurement of Toxicants and Toxic Actions By using a variety of techniques derived from analytical chemistry, bioassays, and applied mathematics, toxicants and their toxic effects can be measured and quantitated. This aspect of toxicology includes a number of important fields (Figure 1.1) as follows: Analytical Toxicology Analytical toxicology is a branch of analytical chemistry that is concerned with methods for the identification and assay of toxic chemicals and their metabolites in biological and environmental samples. Toxicity Testing

Structure-Activity Studies Structure-activity studies are important to understanding of the relationship between the chemical and physical properties of xenobiotics and toxicity and, particularly, the use of such relationships for the prediction of toxicity. Epidemiology Epidemiology, as it applies to toxicology, is closely related to the biomathematical and statistical models and is of great importance since it deals with the study of toxicity as it occurs, rather than in an experimental laboratory setting. 1.3.3 Classification Based on Mechanisms of Toxic Action and Effects Toxicants can also be classified on the basis of all the events leading to exertion of their toxic effects in vivo. This involves studies at the fundamental level of organ, cell, and molecular functions, including their uptake, distribution, metabolism, mode of action, and excretion. Important disciplines include the following: Biochemical Toxicology Biochemical toxicology considers events at the biochemical and molecular level, including enzymes that metabolize xenobiotics, generation of reactive intermediates, and interaction of xenobiotics or their metabolites with macromolecules.

The branch of toxicity testing involves the use of living systems to estimate toxic effects of various chemicals. It covers the entire gamut from short-term tests for genotoxicity such as the Ames test and cell culture techniques to the use of live animals for acute toxicity tests and for lifetime or multigeneration chronic toxicity tests. The term bioassay is used to describe the use of living organisms to quantitate the amount of a particular toxicant present.

Behavioral Toxicology

Biomathematics and Statistics

Nutritional toxicology deals with the effects of diet on the expression of toxicity and the mechanisms for these effects.

Mathematical and statistical techniques are used in a number of areas of toxicology. They deal with data analysis, the determination of significance, and the formulation of risk estimates and predictive models. The latter is particularly important in epidemiology and environmental toxicology.

Behavioral toxicology deals with the effects of toxicants on animal and human behavior. This involves peripheral and central nervous systems as well as effects mediated by other organ systems such as the endocrine glands. Nutritional Toxicology

Genetic Toxicology Genetic toxicology or mutagenesis is concerned with toxic effects of chemicals on the genetic material and the inheritance of these defects.

Toxicological Pathology Toxicological pathology deals with the effects of toxic agents as manifested by changes in subcellular, cellular, tissue, or organ morphological characteristics.


Teratogenesis Teratogenesis includes the chemical and biochemical events that lead to deleterious effects on the developmental

process; it involves multigenerational studies of animals to study the long-term toxic effects on the reproductive processes.

cology, the toxic side effects and testing for them fall within the science of toxicology. Drugs of Abuse

Carcinogenesis Carcinogenesis includes the study of chemical and biochemical events upon exposure to toxic chemicals that lead to the large number of effects on cell growth.

Drugs of abuse are chemicals, often illegal, taken for psychological or other effects that cause dependence and toxicity. Food Additives

Organ Toxicity Organ toxicity considers the effects at the level of organ functions, e.g., neurotoxicity, hepatotoxicity, and nephrotoxicity. Regulatory Toxicology Regulatory toxicology is concerned with the formulation of laws and regulations authorized by laws to minimize the effect of toxic chemicals on human health and the environment. In the United States, the risk assessment, which is the definition of risks, potential risks, and the risk-benefit equations necessary for regulation of toxic substances, is primarily under the control of several government agencies. These include the Food and Drug Administration (FDA), the Environmental Protection Agency (EPA), and OSHA. 1.3.4 Classification Based on Chemical Use Classes

Food additives are of concern to toxicologists only when they are toxic or are being tested for possible toxicity. Industrial Chemicals Because of the sheer number of industrial chemicals used, testing for toxicity and controlling exposure to those known to be toxic constitute a large field of toxicology. Naturally Occurring Substances Naturally occurring substances include many phytotoxins, mycotoxins, and inorganic minerals that occur naturally in the environment and may find a way into the human food chain. Combustion Products Combustion products are generated primarily from fuels and other industrial chemicals.

Hodgson (1987) also classified toxicants on the basis of use classes. This classification includes the toxicological aspects of the development of new chemicals for commercial uses. In some of these use classes, toxicity, at least to some organisms, is a desirable trait; in others, it is an undesirable side effect. This category includes both synthetic and natural products. The following classes can be identified by the use criteria.

1.4

Agricultural Chemicals

1.5

Agricultural chemicals include herbicides, fungicides, pesticides, and rodenticides, in which toxicity to the target organism is a desired quality, whereas toxicity to nontarget species must be prevented. Development of such selectively toxic chemicals is one of the applied roles of comparative toxicology. Clinical Drugs Although the development of clinical drugs is largely the responsibility of the pharmaceutical industry and pharma-


SOURCES OF TOXIC COMPOUNDS

Several natural as well as synthetic compounds are potent toxicants and can enter the human food chain as contaminants. Although toxic, many find important uses at therapeutic dose levels in clinical medicine. Representative sources of toxic compounds are summarized in Table 1.1.

CLASSIFICATION OF TOXICANTS

Toxic agents can be classified in a variety of ways (Klaassen, 1986; Sperling, 1984; Manahan, 1992; Gossel and Bricker, 1984; Hodgson and Levi, 1987). For example, they can be classified in terms of their target organ (liver, kidney, lungs, skin, nervous system, hematopoietic system, etc.), their use (pesticides, solvents, food additives, etc.), their source (animal, plant, microbial toxins), and their effects (cancer, mutation, liver injury, etc.). Toxicants can also be grouped in terms of their physical state (gas, dust, liquid), their labeling requirements (explosive, flam-

Table 1.1 Representative Sources and Examples of Toxic Compounds

Table 1.2

Synthetic organic compounds Air (transportation, industrial processes, electric power generation, heating processes) Carbon monoxide, oxides of nitrogen and sulfur, hydrocarbons, particulates Water (runoffs, sewage, waste products discharged from refineries, swelters, or chemical plants) Agrochemicals, hydrocarbons, detergents, heavy metals Food contaminants Bacterial, fungal, and animal toxins; pesticide residues; plant alkaloids, residues of animal feed additives (e.g., antibiotics, estrogens); industrial chemicals Food additives Nitrates, nitrites Chemicals in the workplace Inorganic metals, aliphatic and aromatic hydrocarbons, halogenated hydrocarbons, alcohols, esters, organometallics, pesticides Drugs of abuse Cocaine, methamphetamines, lysergic acid diethylamide (LSD), morphine, nicotine, barbiturates Therapeutic drugs Essentially all therapeutic drugs, which can be toxic at high doses Agrochemicals Pesticides, herbicides, nematicides, rodenticides Solvents Aliphatic and aromatic hydrocarbons, halogenated solvents, alcohols Polycyclic aromatic hydrocarbons (incomplete combustion of organic materials) Pyrenes, anthracenes Cosmetics Thioglycolates, thioglycerol Naturally occurring toxins Mycotoxins Aflatoxins, fumonisins, ergot alkaloids, tricothecenes, patulin Microbial toxins Tetanus, botulinum, diphtheria, Staphylococcus spp. toxins Plant toxins Ricinine, solanine, chaconine, safrole, quinones, estrogens, enzyme inhibitors, lectins, cyanogenic glycosides Inorganic chemicals Heavy metals, oxides of nitrogen and sulfur

Rating/class

mable, oxidizer), their chemical properties (aromatic amines, polycyclic hydrocarbons, halogenated hydrocarbons, etc.), or their toxic potential, as shown in Table 1.2. According to Klaassen (1986), classification on the basis of the biochemical mechanism of action of toxicants is usually more informative than classification by general


Toxicity Rating Chart

Practically nontoxic Slightly toxic Moderately toxic Very toxic Extremely toxic Supertoxic

Probable lethal oral dose for average human > 15 g/kg 5–15 g/kg 0.5–5 g/kg 50–500 mg/kg 5–50 mg/kg < 5 mg/kg

Source: Gossel and Bricker (1984) and Klaassen (1986).

category, such as irritants and corrosives. Nonetheless, more general classifications, such as air pollutants, occupation-related agents, and acute and chronic poisons, can still provide a useful focus on a specific problem. It is quite evident that no single classification can be used to cover the entire spectrum of toxic agents. In general, classification systems that take into consideration both the chemical and the biological properties of the toxicant and the exposure characteristics tend to provide useful information for legislative or control purposes as well as for toxicology in general.

1.6

FOOD TOXICOLOGY AND THE SCOPE OF THE BOOK

Food toxicology can be defined as a systematic study of toxicants found in foods. These compounds can be of natural origin as products of the metabolic processes of animals, plants, and microorganisms from which the food is derived; as biological and chemical contaminants from the air, water, and soil; as intentionally introduced food additives; and as those formed during the processing of foods. Food toxicology is thus concerned with the toxic potential of food, the conditions and factors affecting the presence of these toxicants in food, their interactions with essential dietary nutrients, the response of the human body to these toxins, and the means of prevention or minimization of these toxic effects as they pertain to food safety and human nutrition. As compared to the presence of toxicants that are naturally present in various foods, biological contamination of our food supply presents grave food safety concerns. Food-borne diseases caused by bacteria and viruses have varying degrees of severity ranging from mild indisposition to chronic or life-threatening illness. Their importance as a vital public health problem is often overlooked because the true incidence is difficult to evaluate and the severity of the health and economic consequences is often

not fully appreciated. For most food-borne diseases, only a small proportion of cases reach the notice of health services, and even fewer are investigated (Kaferstein et al., 1999). It is believed that in industrialized countries less than 10% of the cases are reported, and in developing countries reported cases probably account for less than 1% of the total (WHO, 1984). Studies in some industrialized countries point to an underreporting factor of up to 350 for certain food-borne diseases (Todd, 1989; Norling, 1994). Despite these limitations in reporting, available data give evidence of a tremendous public health problem. Furthermore, even in industrialized countries, the data indicate an increasing trend. Although the situation regarding foodborne diseases is very serious in developing countries, even the industrialized countries have experienced a succession of major epidemics, of which the mad cow disease is the latest entry in a long list of such food-borne outbreaks. The estimated annual incidence of food-borne diseases in the United States ranges from 6.5 million to 80 million cases. Surveys in other countries suggest that up to 10% of the general population may annually suffer from a food-borne disease (Todd, 1989; WHO, 1994; Notermans and Hooenboom-Verdegaal, 1992; Notermans and van de Giessen, 1993). In the United States, typically 400 to 500 food-borne outbreaks are reported annually to the Centers for Disease Control (CDC), with an average of about 40 cases per outbreak, for an average of about 18,000 food-borne disease cases annually (Bean and Griffin, 1990; Bean et al., 1990a, 1990b). However, these data include many fewer cases than do laboratory surveillance data. For example, for 1983 to 1987, 6249 salmonellosis cases per year were reported in the outbreak data compared with 44,000 cases per year in the laboratory surveillance data (CAST, 1994). Bennett and coworkers (1987) estimated that 96% of salmonellosis cases were food-borne. In the developing countries, diarrheal diseases, especially infant diarrhea, are the dominant problem and indeed one of massive proportions. Annually, some 1.5 billion episodes of diarrhea occur in children below the age of 5, and of these over 3 million die as a result (WHO, 1994). Although traditionally contaminated water supplies were believed to be the main source of pathogens causing diarrhea, up to 70% of diarrheal episodes may actually be due to food-borne organisms (Esrey and Feachem, 1989; Motarjemi et al., 1993). In addition to the food-borne diseases, food contamination with mycotoxins, pesticide residues, drug residues, and industrial chemicals is a serious issue that affects human safety and well-being. It should, however, be noted that such contamination occurs on a sporadic basis. Furthermore, it can easily be prevented by using


careful food production, storage, handling, and preparation practices. The primary aim of writing this book is to provide comprehensive information on the chemical and toxicological characteristics of various toxicants that occur in the human food chain in a single volume. Part I presents information on the basic concepts of toxicology as a science. Here, general information is presented on the basic principles of toxicology, the chemical and biochemical basis of toxicity of chemicals, manifestations of toxic effects, carcinogenesis, and detoxification mechanisms. Information on food toxicants from various sources is presented in Part II. Although these food toxicants are described individually and may indeed appear to present grave dangers to human health when consumed, the readers should be aware of the fact that any treatment of food toxicology must be considered from the viewpoint of overall nutrition. This is primarily because, as described earlier, there is no such thing as “absolute safety,” and everything we consume, from water to salt, sugar, proteins, and fats, has some associated toxicity. Moreover, toxic effects of these compounds are generally not additive. We thus consume many toxic substances in our normal diet without showing any signs of intoxification. Furthermore, several compounds thought to be antinutritional or toxic in the 1970s and early 1980s have now shown to have beneficial effects on human nutrition and well-being. In fact, if it were not for the biological contamination of foods and food hygiene issues, we probably now enjoy a safer food supply in a wider variety of forms than at any other time in the history of human civilization. The author would like to bring out this important message in this book.

REFERENCES Bean, N.H. and Griffin, P.M. 1990. Foodborne disease outbreaks in the United States, 1973–1987: Pathogens, vehicles, and trends. J. Food Protect. 53:804–817. Bean, N.H., Griffin, P.M., Goulding, J.S., and Ivey, C.B. 1990a. Foodborne diseas e outbreaks, 5-year summa ry, 1983–1987. Mor. Mortal. Wkly. Rep. CDC Surveill. Summ. Morb. Mort. Weekly Rep. (MMWR) 39(SS1):15–59. Bean, N.H., Griffin, P.M., Goulding, J.S., and Ivey, C.B. 1990b. Foodborne diseas e outbreaks, 5-year summa ry, 1983–1987. J. Food Protect. 53:711–728. Bennett, J.V., Holmberg, S.D., Rogers, M.F., and Solomon, S.L. 1987. Infectious and parasitic diseases. In Closing the Gap: The Burden of Unnecessary Illness, eds. R.W. Amler and H.B. Dull, pp. 102–114. Oxford University Press, New York.

Borlaug, N.E. 1981. Using plants to meet world food needs. In Future Dimensions of World Food and Population, ed. R.G. Woods, pp. 101–153. Westview Press, Boulder, CO. CAST. 1994. Foodborne pathogens: Risks and consequences. Task Force Report No. l22. Council for Agricultural Science and Technology, Ames, IA. Deshpande, S.S. 1992. Food legumes in human nutrition: A personal perspective. CRC Crit. Rev. Food Sci. Nutr. 32:333–363. Doull, J. and Bruce, M.C. 1986. Origin and scope of toxicology. In Toxicology: The Basic Science of Poisons, 3rd ed., eds. C.D. Klaassen, M.O. Amdur, and J. Doull, pp. 3–10. Macmillan, New York. Esrey, S.A. and Feachem, R.G. 1989. Interventions for the Control of Diarrheal Diseases Among Young Children. Promotion of Food Hygiene. World Health Organization, Geneva. Garfield, E. 1990. Journal citation studies. 53. Agricultural sciences: Most fruitful journals and high yield research fields. Curr. Contents 21(51):3–10. Gossel, T.A. and Bricker, J.D. 1984. Principles of Clinical Toxicology. Raven Press, New York. Hodgson, E. 1987. Introduction to toxicology. In Modern Toxicology, eds. E. Hodgson and P.E. Levi, pp. 1–22. Elsevier, New York. Hodgson, E. and Levi, P.E. 1987. Modern Toxicology. Elsevier, New York. Holmstedt, B. and Liljestrand, G. 1981. Readings in Pharmacology. Raven Press, New York. Kaferstein, F.K., Motarjemi, Y., Moy, G.G., and Quevado, F. 1999. Food safety: A worldwide public issue. In International Food Safety Handbook, eds. K. van der Heijden, M. Younes, L. Fishbein, and S. Miller, pp. 1–20. Marcel Dekker, New York.


Klaassen, C.D. 1986. Principles of toxicology. In Toxicology: The Basic Science of Poisons, 3rd ed., eds. C.D. Klaassen, M.O. Amdur, and J. Doull, pp. 11–32. Macmillan, New York. Loomis, T.A. 1978. Essentials of Toxicology. 3rd ed. Lea & Febiger, Philadelphia. Manahan, S.E. 1992. Toxicological Chemistry. Lewis Publishers, Boca Raton, FL. Motarjemi, Y., Kaferstein, F., Moy, G., and Quevado, F. 1993. Contaminated weaning food. A major risk factor for diarrhea and associated malnutrition. Bull. World Health Organ. 71(1):79–92. Norling, B. 1994. Food poisoning in Sweden: Results of a field study. Report No. 41/94. National Food Administration, Uppsala, Sweden. Notermans, S. and Hooenboom-Verdegaal, A.S. 1992. Existing and emerging foodborne diseases. Int. J. Food Microbiol. 15:197–205. Notermans, S. and van de Giessen, K. 1993. Foodborne diseases in the 1980s and 1990s. Food Control 4(3):122–124. Sperling, F. 1984. Toxicology: Principles and Practice. John Wiley, New York. Todd, E.C.D. 1989. Preliminary estimates of costs of foodborne diseases in Canada and costs to reduce salmonellosis. J. Food Protect. 52:586–594. WHO. 1984. The role of food safety in health and development: A report of a Joint FAO/WHO Expert Committee on Food Safety. Technical Report Series No. 705. World Health Organization, Geneva. WHO. 1994. Program for Control of Diarrheal Diseases. 9th Program Report 1992–1993. World Health Organization, Geneva. Wittwer, S.H. 1980. The shape of things to come. In The Biology of Crop Productivity, ed. P.S. Carlson, pp. 413–436. Academic Press, New York.

2 Principles of Toxicology

2.1

INTRODUCTION

A poison or toxicant is a chemical that is harmful to living organisms because of its detrimental effects on tissues, organs, or biological processes. Any chemical may be a poison at a given dose and route of administration. Three factors primarily influence the toxicity of any chemical to a given species: the toxic substance itself and the matrix in which it is present, the circumstances of exposure, and the organism and its environment. Usually, an experimentally determined acute oral toxicity expression, such as an LD50 value, which is the dose required to kill half of test subjects, is used to express the toxic potential of any given chemical. Such estimates, however, are not an absolute description of the compound’s toxicity in all individuals or across different species. They neither assess the inherent capacity of the compound to produce an injury nor reflect the victim’s ability to respond in a manner other than predicted. Hence, quantitative estimates of toxicity in terms of mortality are usually not good parameters for toxicity measurements. Much more widespread than fatal poisoning, and certainly more subtle, are various manifestations of morbidity or unhealthiness. Morbidity can be manifested in several ways. Whereas the effects on vital signs are obvious, it is the subtle effects that are not life threatening per se but nonetheless are responsible for minor health ailments that ultimately cost millions of dollars in terms of treatment expenses and loss of productivity. In some instances, a toxic response may not be observed for years. It is therefore essential to distinguish acute toxicity, which


has an effect soon after exposure, and chronic toxicity, which has a long latency period. In practical situations, therefore, the critical factor is not the intrinsic toxicity of a chemical, but rather the risk or hazard associated with its use. In food science and nutrition, it is especially important to understand the concepts of relative risks and safety, hazard, and toxicity associated with the consumption of foods. Risk is the probability that a substance will produce harm under specified conditions. Absolute safety, in contrast, is the assurance that damage or injury from use of a substance is impossible. However, as discussed in Chapter 1, absolute safety is virtually unattainable. Hence, the concept of relative safety has been proposed (Hall, 1988, 1991). Relative food safety then can be defined as the practical certainty that injury or damage will not result from the consumption of food or ingredients used in food processing in a reasonable and customary manner and quantity. Food safety, however, does not refer to the food itself, but also to the people consuming it. For example, foods considered safe for most people when used in a reasonable and customary manner and quantity can be extremely toxic, even lethal, to certain sensitive or allergic individuals. The concepts of toxicity and hazard are also relevant to any discussions of food safety. Toxicity, as defined earlier, is the capacity of a chemical to produce harm or injury of any kind (chronic or acute) under given conditions. Generally, for humans, any deviation from normal is considered as a possible negative effect, even though the change may seem to be positive, such as increased growth

rates or enhanced nutrient absorption. The change is assumed to be negative until proved beneficial. Hazard is the relative probability that such harm or injury will result when the substance is used in a proposed manner and quantity. Assessments of whether a food or ingredient is safe should not be based on its inherent toxicity alone, but on whether or not a hazard is created. The inability to distinguish between toxicity and hazard as associated with the consumption of foods, especially by the general public, often results in inaccurate assessments of the relative risks and safety of our food supply. Both the regulations and the way that they are applied by the regulatory agencies often reflect the public attitudes toward particular types of chemicals and specific kinds of risks. The way that people generally perceive risk is quite different from the way scientists analyze risk, and this dichotomy has led to conflicts in the public policy arena. Generally, people tend to view catastrophic risks, e.g., airplane crashes, as greater than ordinary ones, e.g., automobile accidents. Similarly, synthetic chemicals are often viewed as riskier than natural ones; voluntary risks, such as smoking, as less significant than involuntary ones, such as air pollution; and those with immediate effects as less of a risk than those with delayed effects. Some of these factors are summarized in Table 2.1. The question of what constitutes an acceptable risk is also a matter of judgment. Such decisions are multifaceted and complex and involve a balance of risk and benefit. High risks may be acceptable in the use of lifesaving drugs but be unacceptable for food additives. Klaassen (1986a) has suggested the following factors that must be considered in determining an acceptable risk: •

Table 2.1

Benefits gained from use of the substance

Public Perception of Risk

Criteria Origin Volition Effect manifestation Severity (number of people affected per incident) Controllability Benefit Familiarity Exposure Necessity

Characteristics perceived as lower risk

Characteristics perceived as higher risk

Natural Voluntary Immediate Ordinary

Synthetic Involuntary Delayed Catastrophic

Controllable Clear Familiar Continuous Necessary

Uncontrollable Unclear Unfamiliar Occasional Luxury

Source: Kamrin (1988).


• • • • • •

Adequacy and availability of alternative substances to meet the identified use Anticipated extent of public use Employment considerations Economic considerations Effects on environmental quality Conservation of natural resources

Public perception apart, several biological factors modify the response of a species to the toxic agent. Realization of these factors is intrinsic to our fundamental understanding of the principles of toxicology. This forms the basis of this chapter. For a more detailed treatment of the subject, the readers are advised to consult several excellent textbooks and monographs (Loomis, 1978; Hodgson and Guthrie, 1980; Timbrell, 1982; Gossel and Bricker, 1984; Klaassen et al., 1986; Hodgson and Levi, 1987; Kamrin, 1988; Marquis 1989; Manahan, 1992; Stine and Brown, 1996; Niesink et al., 1996).

2.2

TYPES AND CIRCUMSTANCES OF EXPOSURE

The exposure of an organism to a toxic substance is of prime importance in toxicology. In this regard, both the duration of exposure per incident as well as the frequency of exposure need to be considered. The rate of exposure, inversely related to the duration per exposure, and the total period over which the organism is exposed are primarily situational variables. Other factors that affect the toxicity of a substance include the dose, toxicant concentration, the exposure site, and the route of absorption. It is important to understand the differences between acute and chronic poisonings or exposures and the acute and chronic effects or symptoms. Acute and chronic poisonings differ in the number and duration of exposures to the toxicant. The toxic effects manifested as a result of poisoning may be either local, i.e., confined to a specific tissue or organ, or systemic. Generally, acute local exposure occurs at a specific location as single exposure to the toxicant. It may occur over a period of few seconds to a few hours and may affect the exposure site, particularly skin, eyes, or mucous membranes. Similar tissues or organs can also be affected by chronic local exposure. However, the time span for the manifestation of toxic effects could be several months or even years. As compared to local exposures, systemic exposures are usually manifested in toxic symptoms or effects in tissues or organs that are remote from the entry site. Thus toxicants may enter the body by inhalation or ingestion and affect organs such as the liver. The acute and chronic

systemic poisonings primarily differ in that the exposure occurs over a prolonged period in the latter case. It is also likely that an acute exposure or poisoning may result in chronic symptoms. Thus, a single exposure to potent carcinogens, such as aflatoxins or nitrosamines, may result in the chronic symptom of cancer, whereas a chronic exposure to cyanide in sufficient dose always results in acute symptoms. Therefore, the terms acute and chronic, when used to describe symptoms, refer to the duration and reversibility of the symptoms. An acute symptom is of short duration, usually severe, and generally reversible after removal of the toxicant. Chronic symptoms, in contrast, are prolonged and persist even after removal of the toxicant. For example, some organophosphate pesticides can produce chronic paralysis (Hays, 1972). Liver carcinogens in small doses may result in hepatic cancer but acute hepatic damage in large doses. Generally, exposures to toxicants between 24 hr and 90 days are usually referred to as prolonged intoxication (Concon, 1988). The symptoms of exposure or poisoning may be immediate or delayed. They are influenced by factors such as dose, type of compound, and route of contact. Toxic compounds, which produce immediate symptoms, may be easily identified as the cause of intoxication and, therefore, can be avoided. In contrast, those with delayed symptoms, especially if the effects do not appear until after several months or years, are not easily identified. Many food poisons fall under the highly delayed category. These compounds, therefore, are of concern because of their possible role in modern epidemic diseases whose causes have been difficult to establish. Furthermore, when symptoms are delayed, antidotal therapy, assuming that it is known, may be difficult to administer unless the symptoms have been correlated with those of a particular toxicant. Even so, antidotes are of little value when the appearance of symptoms, such as cancer and other effects indicating structural tissue damage, is markedly delayed. Although chronic symptoms are not necessarily due to the accumulation of the poison or toxicant in the tissues, there are toxicants that accumulate in the tissues to toxic levels, resulting in chronic delayed toxic symptoms. Some well-known examples of such toxicants include heavy metals, such as lead and mercury, and organochlorine pesticides such as dichlorodiphenyltrichloroethane (DDT). Similarly, some toxicants do not elicit chronic poisoning. For example, many of the neurotoxins that are extremely powerful poisons, e.g., the botulinum exotoxin, may progress from the no effect level to the lethal level without passing through the chronic range. In contrast, several carcinogens at the lower doses initiate carcinogen-


esis but produce acute hepatic or renal damage when administered at high doses. 2.2.1 Exposure Assessment Because the problem of chronicity has significant bearing on public health, it is essential that those compounds having this property be identified and their other toxicological properties established. Therefore, some quantitative measure of the chronicity of a compound is important. However, the determination of chronicity has the inherent weakness in that it is based on data obtained from experimental animals with short life spans. Therefore, a major problem of validly extrapolating such data to humans remains. Nevertheless, such indices of chronicity may be potentially useful in serving as a guide in identifying those compounds, which may behave similarly in humans. Defining the exposure to a toxic agent is thus essential in assessing the significance of toxicological tests as well as understanding the risks to both humans and other organisms. There is no one uniform, well-established procedure for expressing exposure. Some commonly used expressions are summarized in Table 2.2. For proper risk assessment, the units in which exposure is expressed must be compatible with those that toxicologists commonly use to report the results of their experiments. The toxic pattern of exposure is also critical. To interpret the toxicological significance of an exposure, the following factors need to be considered (Brown and Bomberger, 1982; Brown, 1987): 1. 2. 3. 4.

Table 2.2 Toxicants

Duration of each exposure, if not instantaneous Frequency with which the exposure is repeated Variation of exposure level within each period of exposure Elapsed time to the observation of effects from the last (or sometimes first) exposure period

Methods of Expressing Exposure to Chemical

Method Concentration in medium

Quantity available for absorption

Rate of intake or exposure Concentration in body tissue Organ dose

Units mg/kg Food mg/L Water mg/m3 Air mg Inhaled, total mg Inhaled/kg body weight mg Ingested, total mg Ingested/kg body weight mg/kg Body weight mg/ml Serum mg To liver

Several possible time patterns of exposure of an individual to a toxicant are shown in Figure 2.1. The shortterm averages are generally appropriate for assessing acute toxicity responses, whereas the long-term annual average might be more appropriate for assessing chronic toxicity, such as carcinogenic or mutagenic potential of a given toxicant. The latter is especially important when a linear dose-response relationship is seen. Finally, for the control of various sources of exposure for risk assessment analysis, one needs to determine how much exposure is derived from each source. In this regard, the following factors need to be defined for a complete characterization of exposure to any given toxicant: • • • • • •

Specification of levels of exposure Specification of route(s) of exposure Distribution of exposures over time Distribution of exposures over geographical region Distribution of exposure over segments of population at risk Distribution of exposures over various sources

Detailed information on the methodology used for assessing the exposure to toxicants and the accompanying risk-benefit/safety analysis is supplied in several reviews (Brown and Bomberger, 1982; Brown and Suta, 1982; Klaassen, 1986a; Brown, 1987; Kamrin, 1988). An excellent monograph is also available on the principles of data interpretation including the statistical techniques used for such purposes (Tardiff and Rodricks, 1987).

Figure 2.1 Time patterns of exposure to toxicants: A, continuous; B, intermittent; C, cyclic; D, random; E, concentrated. (Adapted from Brown and Bomberger [1982].)


2.3

ROUTES OF TOXICANT EXPOSURE AND ABSORPTION

To exert a toxic effect, a compound must have contact with the biological system under consideration. The toxicant may exert a local effect at the site on initial exposure, but it must penetrate the organism in order to have a systemic effect. In fact, one of the main factors that influence the dose at the site of action, or the effective dose, is the route of exposure or the way in which the individual was exposed. Furthermore, the manner by which a potentially toxic chemical is introduced into the body can influence the time of onset, intensity, and duration of the toxic effects. The route of exposure may also predict the degree of toxicity and possibly the target systems that are most readily affected. Chemicals may be introduced into the complex biological organisms by a variety of routes. The major routes of accidental or intentional exposure to toxicants of humans and other animals include the skin (percutaneous route), the lungs (inhalation, respiration, pulmonary route), and the mouth (ingestion, oral route) (Figure 2.2). Other minor routes of exposure include rectal, vaginal, and parenteral. The parenteral route, viz., intraperitoneal, intramuscular, intravenous, or subcutaneous) is primarily confined to the administration of therapeutic agents. The chemical and physical properties of each compound largely determine the route by which intentional or accidental exposure occurs. The pulmonary system is most likely to take in toxic gases or very fine, respirable solid or liquid particles. In other than a respirable form, a solid usually enters the body orally. The percutaneous or dermal route is important in the absorption of liquids, solutes in solution, and semisolids through the skin. The site of entry of a toxicant is an important factor in the manifestation of final toxic effects. Thus, compounds taken by the oral route may be hydrolyzed to less (or sometimes more) toxic metabolites when exposed to the acid conditions in the stomach. The intestinal microflora may also change the nature of the compound by metabolism and thereby affect the toxicity outcome. The site of entry is also important to the final disposition of the compound. Thus, absorption through the skin may be slow and result in initial absorption into the peripheral circulation. Absorption from lungs, in contrast, is generally rapid and exposes major organs very quickly. Compounds absorbed from the gastrointestinal (GI) tract after oral exposure first pass through the liver, where they may be extensively metabolized. Irrespective of the route of exposure and absorption, there is always a potential for the toxicant to be absorbed into the bloodstream. If this occurs, the toxicant is then transported throughout the body,

Figure 2.2

Major sites of exposure, metabolism and storage, routes of distribution, and elimination of toxic substances in humans.

thereby potentially exposing all the organs and tissues. If one or more of these parts of the body are more sensitive than the site of entry, more severe toxic effects may occur to that organ or tissue. Furthermore, if the toxicant or its metabolites remain in the blood circulation for a long period, the tissues and organs are exposed to them repeatedly. Thus a single external exposure may lead to repeated internal exposures and possibly toxicity to a number of organs and tissues. Once the toxicant is absorbed and enters the bloodstream, it may undergo several metabolic changes. For example, it may be excreted or stored in body tissues as is, or it may interact with other body chemicals and be altered in some way. In the latter instance, since the metabolism is not 100% efficient, one or more metabolites may be generated from the parent toxic compound. All of the metabolites then undergo fates similar to that of the original absorbed material. Thus exposure to one chemical may result in the excretion or storage of several different chemicals as well as the potential for a variety of toxic effects. It should be emphasized that these are usually not either/or possibilities—the absorbed substances and their metabolites are often partially excreted, partially stored, and partially available to produce adverse effects (Kamrin, 1988). As a general rule, a chemical or toxicant injected by the intravenous parenteral route would be expected to be the most toxic. Administered by other routes, the approximate descending order of toxicity would be inhalation > intraperitoneal > subcutaneous > intramuscular > intrader-


mal > oral > topical. Of course, not all of these routes are important in food toxicology. Nonetheless, the salient features of various routes of toxicant exposure and absorption are briefly described in the following sections. However, because of its obvious importance in food toxicology, a major emphasis is placed on the oral route of toxicant absorption involving the GI tract. 2.3.1 Percutaneous Exposure The simplest and most common exposure of humans and animals to exogenous chemicals of all types is exposure through accidental or intentional contact of the chemical with the skin. Toxicants can enter the skin through epidermal cells, sebaceous gland cells, or hair follicles. By far the greatest area of the skin is composed of the epidermal cell layers, and most toxicants absorbed through the skin enter through epidermal cells. Despite their much smaller total areas, however, the cells in the follicular walls and in sebaceous glands are much more permeable than epidermal cells (Manahan, 1992). The skin is a complex, multilayered tissue comprising approximately 19,000 cm2 of surface in an average human and contributing approximately 10% of the body weight (Guthrie and Hodgson, 1987a). It is a membrane that is relative impermeable to most ions as well as aqueous solutions. However, it is permeable to a large number of toxicants in the solid, liquid, or gaseous phase.

Although skin is the most readily accessible organ to all forms of chemicals, it is also an efficient barrier to most environmental toxins. The major barrier to dermal absorption of toxicants is the stratum corneum, or the outermost horny layer composed of highly keratinized cells. The permeability of the skin is inversely proportional to the thickness of this layer. It varies by location in the body in the order soles and palms > abdomen, back, legs, arms > genital (perineal) area. Disruption of the stratum corneum essentially removes all but a superficial deterrent to penetration, since the two other main areas of skin, viz., the dermis and the subcutaneous tissue, offer little resistance to penetration. Therefore, breaks in epidermis due to laceration, abrasion, or irritation increase the permeability, as do inflammation and higher degrees of skin hydration. However, in order to reach systemic circulation, the toxic compound still has to traverse several layers of cells; in contrast, in the GI tract, only two cell layers separate the toxicant from the bloodstream. Compounds, which are well absorbed percutaneously, are generally very lipid-soluble. In general, gases penetrate quite freely through the epidermal tissues, liquids less freely, and solids that are insoluble in water probably are incapable of penetrating to a significant degree. Penetration of a toxicant via the percutaneous route is also time dependent and a function of concentration gradient.

2.3.3 Oral Route The oral route is a major site of entry into the body for many toxic compounds. Food additives, food toxins, licking or rubbing, and airborne particles excluded from passage to the alveoli and returned to the glottis are among potential avenues for accidental ingestion. The GI tract can be regarded as a tube through the body from the mouth to the anus (Figure 2.3). Although it is within the body, its contents are essentially exterior to the body fluids. Therefore, toxicants in the GI tract can produce an effect only on the surface of the mucosal cells that line the tract. Any systemic effect of toxicant ingested by the oral route, therefore, requires its absorption through the mucosal cells that line the inside of the GI tract. Toxicants can be absorbed throughout the GI tract, including the buccal cavity and rectum. However, because of short residence times, most substances are not readily absorbed in the mouth or esophagus. The stomach is the first part of the GI tract where substantial absorption and translocation to other parts of the body may take place. Because of the stomach’s low pH (about 1.0), absorption through stomach is dependent on the amount of nonionized form available. Therefore, substances that are weak

2.3.2 Pulmonary/Inhalation Route The pulmonary system is the site of entry for numerous toxicants. Absorption via the lungs is an important route for toxic gases, volatile solvents, and aerosols and, in some cases, airborne particles. The pulmonary route can greatly accentuate the expected onset of toxicity for a given compound for two reasons. The rich capillary exchange at the deeper lung recesses causes a toxicant at the lung surface to be separated by only 1–2 µm from the circulation, enabling exchange of gases to occur in seconds or less. In addition, the surface area of lungs (about 50–100 m2) is some 50 times the area of skin. Because of its unpreventable contact with contaminated air, the respiratory system has also developed numerous mechanisms to avoid many airborne substances. Particles can be trapped in the upper respiratory or nasopharyngeal region. Those deposited in the tracheobronchiolar region are cleared upward by the mucus blanket. In addition to upper pathway clearance, lung phagocytosis is very active in both upper and lower pathways of the respiratory tract and may be coupled to the mucus cilia. Phagocytosis may also direct engulfed toxicants into the lymph.


Figure 2.3 Schematic diagram of the alimentary canal and associated structures in the human. A, parotid gland (salivary gland); B, pharynx; C, sublingual gland (salivary gland); D, submandibular gland (salivary gland); E, esophagus; F, cardiac sphincter; G, stomach; H, liver; I, gallbladder; J, pancreas; K, duodenum; L, splenic flexure of transverse colon; M, hepatic flexure; N, transverse colon; O, descending colon; P, ascending colon; Q, jejunum; R, caecum; S, appendix; T, ileum; U, sigmoid colon; V, rectum; W, anus.

acids (i.e., ionic at pH near 7.0 and above) are neutral in stomach so that they readily traverse the stomach walls. In some cases, absorption is affected by stomach contents other than HCl. These include food particles, gastric mucin, gastric lipase, and pepsin. Compared to gastric absorption, intestinal absorption is extensive because of the presence of microvilli, which provide an extremely large surface area (Figure 2.4). The pH of the contents of the small intestine is close to neutral, so that weak bases that are charged in the acidic environment of the stomach are uncharged and absorbable in the intestine. Intestinal contents are moved through the intestinal tract by peristalsis, which has a mixing action on the contents and enables absorption to occur along the length of the intestine. There is also significant absorption of compounds in the colon. The epithelium of the colonal lumen behaves much as the small intestine does. The colon is also the major site of water absorption. Thus, the dragging effect is probably highly operative in this part of the GI tract. Furthermore, the metabolic activity of the colon microflora may change the absorbability as well as the toxicological properties of a compound. Their effects in transforming the acidity of this organ’s secretion as well as the acid-base and lipid solubility properties of the compound itself by metabolic degradation may transform the toxicological characteristics of the food altogether. Toxicants ingested by the oral route can also enter the bloodstream via the enterohepatic circulation system (Figure 2.2), which comprises the intestine-blood-liverbile loop. A toxicant absorbed through the intestine goes either directly to the lymphatic system or to the portal circulatory system. The latter carries blood to the portal vein

Figure 2.4 Schematic drawing of the lining of the small intestine. LP, lamina propria.


that goes directly to the liver. The liver serves as a screening organ for xenobiotics, subjecting them to metabolic processes that usually reduce their toxicities, and secretes these substances or their metabolites back to the intestine. For some toxicants, there are mechanisms of active excretion into the bile in which the substances are concentrated by one to three orders of magnitude over their levels in the blood. Other substances enter the bile from blood simply by diffusion. The importance of enterohepatic circulation is discussed in greater detail in the section dealing with the excretion of toxicants in this chapter. The presence and type of food in the stomach can modify the absorption of a toxicant. A meal rich in protein or fat usually delays absorption. Carbonated beverages increase the rate of intestinal absorption by increasing gastric emptying time, with evolution of carbon dioxide. Ingestion of a concentrated chemical frequently causes a decrease in absorption as a result of gastric irritation and constriction of the pyloric sphincter. On the positive side, the oral route of intoxication may provide the body with a chance to metabolize the ingested toxicant readily.

2.4

MECHANISMS OF TOXICANT ABSORPTION

The toxicity of a chemical is dependent on the dose administered. However, it refers not to the dose administered, but rather to the concentration of the toxic chemical in the target organ. To exert its toxic action, a chemical must be absorbed in the biological tissue or organ. If the fraction of the chemical absorbed is low or the rate of absorption is low, then only a low concentration of the chemical in the target organ may be obtained, resulting in little or no toxicity. In this section, various mechanisms of toxicant absorption are briefly described. Again, a special emphasis is placed on the mechanisms of intestinal absorption. A toxicant may pass through a number of barriers before achieving a sufficient concentration in the organ where it produces its characteristic toxic effects. These include membranes of a number of cells. In all cases, the membranes of tissue, cell, and cell organelle are basically similar. They comprise a bimolecular layer of lipid molecules coated on each side with a protein layer. Certain branches of the protein layer appear to penetrate the lipid bilayer, and others extend through the membrane. The lipid portion of the membrane consists primarily of phosphatidylcholine, phosphatidylethanolamine, and cholesterol. The fatty acids of the membranes do not have a rigid crystalline structure and at physiological temperatures are quasi-fluid in character. The fluidity of

the membranes is largely determined by the structure and relative proportion of unsaturated fatty acids. When the membranes contain more unsaturated fatty acids, they are more fluidlike, and active transport (defined later in this section) is more rapid (Klaassen, 1986a; Guthrie and Hodgson, 1987a). The mechanism of the movement of toxicants across membranes is a poorly researched area. A toxicant may pass through a membrane by one of two general processes (Klaassen, 1986a): diffusion or passive transport of the chemical, in which the cell expends no energy in its transfer, and specialized transport, in which the cell takes an active part in the transfer of the toxicant through the cell membranes. In spite of these processes, the intestinal mucosae are relatively impermeable tissues to many substances, including various electrolytes, many organic compounds, and water-soluble macromolecules such as starches, pectins, and other heteropolysaccharides and hydrocarbons (Crane, 1979; Henning, 1979; Concon, 1988). 2.4.1 Passive Transport Most toxicants cross membranes by simple diffusion. Simple diffusion of compounds with appropriate water/lipid partition coefficients largely determines the rate of toxicant movement. However, the nature of the mucosal membrane is such that even passive or simple diffusion of compounds is selective. Six factors primarily govern the passive diffusion of substances: (a) Fick’s law, (b) molecular size, (c) lipid solubility, (d) degree of ionization, (e) “drag” effect or bulk flow of the absorption of water, and (f) the Donnan distribution effect. Their relationship to the movement of toxicants across the membrane barriers is briefly described in the following sections. Fick’s Law Many small hydrophilic compounds diffuse across cell membranes through aqueous channels. For molecules that can easily pass through these pores, Fick’s law can predict their rate of diffusion J = KA (Ce – Ci)/h = PA (Ce – Ci) Where

P

= K/h, permeability coefficient

Fick’s law holds quite well for nonelectrolytes such as urea. However, an additional mechanism is also operative with substances such as fructose, mannose, and xylose, even though their absorption also appears to follow Fick’s law. These sugars appear to be absorbed by facilitated diffusion. Simple diffusion through the cell membrane obeying Fick’s law generally cannot take place against a concentration gradient and is not inhibited by metabolic inhibitors. Furthermore, there is no competitive absorption with other substances (Schanker, 1961; Levine, 1970; Concon, 1988). Molecular Size The molecular size of a chemical also influences the rate of intestinal absorption. Generally, an inverse relationship is observed as the rate of absorption decreases with increasing molecular size. However, several factors influence the effect of molecular size on the absorption of these compounds. In this regard, lipid solubility and ionization effects are more important in regulating the passive absorption of compounds through the intestinal tract. Lipid Solubility Some substances seem to be absorbed through the intestinal tract by passive diffusion even though their molecular size greatly exceeds the postulated average pore size of the intestinal epithelium. Thus a route of entry through the cell membrane other than through the aqueous channels must be available to these molecules. These substances enter the cell by “dissolving” into the cellular membrane material, which is highly lipophilic in nature. Thus, their rate of absorption can be correlated to their solubility in oil or lipid solvents. Often, a linear relationship between lipid solubility and absorbability is seen for compounds with similar chemical properties. However, such correlation is often poor for compounds that, in addition to their lipid solubility characteristics, also behave as weak electrolytes in aqueous medium. The rate of absorption of such compounds through the cell membranes can be markedly affected by a change in pH as described later. Degree of Ionization

J

= rate of diffusion

k

= diffusion coefficient

A

= area of surface diffusion

h

= membrane thickness

Ce, Ci = concentrations of the solute outside and inside the cell, respectively


Many toxic chemicals exist in solution in both ionized and nonionized forms. The ionized form is often unable to penetrate the cell membrane because of its low lipid solubility, whereas the nonionized form may be sufficiently lipid-soluble to diffuse across the cell membrane. Diffusion is thus primarily dependent on the lipid solubility of the nonionized form of the compound. This phenomenon

is observed with a wide range of chemicals, including weak acids and ammonium salts, and dyes. This is particularly true of chemicals for which absorption is not also mediated by mechanisms other than simple diffusion. The degree of ionization of a compound, i.e., the ratio of ionized and nonionized species (A–/HA), can be calculated from the Henderson-Hasselbalch equation pH = pKa + log (A–/HA) where A– and HA are the ionized and nonionized species, respectively. The amount of weak organic acid or base in the nonionized form is dependent on its dissociation constant. The pH at which an acid is 50% dissociated (ionized = nonionized) is called its pKa. It is defined as the negative logarithm of the acid dissociation constant. Conventionally, the dissociation constant for both acids and bases can be expressed as a pKa. The degree of dissociation and ionization of a weak acid or base is dependent on the pH of the medium. This relationship for a weak acid, benzoic acid, and a base, aniline, is shown in Figure 2.5. As the pH decreases, more of the acid becomes nonionized. The converse is true as the pH is increased. For an organic base like aniline, the opposite occurs. Thus, weak acids with pK a values of around 2 are ionized in the small intestine at pH 6 to the extent of approximately 10,000:1, and with weak bases with pKa of around 9, the degree of dissociation is around 1000:1. Since the effective mucosal cell pH is around 5, Schanker and coworkers (1958) postulated that for weak

Figure 2.5 Effect of pH on the ionization of a weak organic acid, benzoic acid (pKa = 4, curve A), and a weak organic base, aniline (pKa = 5, curve B).


acids with a pKa of 2, the degree of dissociation in the cell is around 1000:1, whereas that of weak bases with pKa values around 9 is about 10,000:1. These values appear to be the minimal ratios that must be present for effective absorption of these compounds in the GI tract. Since the lipid soluble form (nonionized) of a weak electrolyte is the species that crosses cell membranes, organic acids are more likely to diffuse across membranes when they are in an acidic environment, whereas a basic environment favors diffusion of bases across membranes. Thus, on the basis of the effect of pH in the absorption of compounds, the degree of toxicity of a weak acid or base may be predicted from the pH of the GI tract. However, when the total area of absorptive surface and the rate of passage are considered, the pH effect may not have a significant influence on the total amount absorbed. For example, the absorption of a weakly acidic substance may be highly favored in the stomach as the pH of its contents decreases, but more of the substance may be absorbed in the intestines, assuming a normal rate of emptying occurs. This is because even though greater dissociation occurs in the intestines, the pH here permits the existence of a significant proportion of nonionized species, and the large surface area compared to that of stomach results in greater total absorption. Thus, even though the rate of absorption in the intestines per unit area may be less than that in the stomach, the greater total surface area of the former results in greater total absorption. Therefore, the large surface area of the intestines may obviate any advantage or disadvantage that the ionization effect may have in the absorption of chemicals. The ionizing effects also explain the poor absorbability by passive diffusion in the small intestines of salts, strong acids, bases, and organic and essential cations such as Fe2+, Fe3+, Mg2+, Ca2+, Zn2+, Mn2+, Co2+, and other trace metallic ions, and the anions, such as PO43– and Cl–. Examples of organic anions that are poorly absorbed are citrates, lactates, and tartrates. The poor absorbability of these substances, in fact, forms the physiological basis for their cathartic effects. Similarly, nonabsorbable sulfates and phosphates are also cathartics. Therefore, for nutritionally essential minerals, special mechanisms are necessary for absorption in order to provide adequate amounts for the animal’s nutritional requirements. However, even with these special mechanisms, absorption of these minerals is limited and is controlled by the body’s physiological requirements. These limitations afford a measure of protection for the organism because above a certain level, these substances are quite toxic (Ulmer, 1977; Concon, 1988). The mechanisms involving active or carrier-mediated transport are described later in this section.

“Drag” Effect and Bulk Flow of the Absorption of Water

Donnan Distribution Effect

When water flows in bulk across a porous membrane, any solute that is small enough to pass through the pores flows with it. Passage through these channels is called filtration, since it involves bulk flow of water due to a hydrostatic or osmotic force. Because such aqueous channels in most cells are relatively small (4–10 nm), only chemicals with molecular weight of 100 to 200 daltons can pass through (Schanker, 1961, 1964). Larger molecules are excluded except in more highly porous tissues (approximately 70-nm pore size) such as kidneys and liver. These allow molecules smaller than albumin (molecular weight 68,000) to pass through. Because many toxicants are relatively large molecules, this pathway is often of limited importance. The bulk flow of absorption of water follows Poiseuille’s law:

A pH differential on each side of a membrane allows only undissociated particles to diffuse out. The process, termed the Donnan distribution effect, causes a net transfer of a chemical to another compartment. Thus, an ionizable chemical that is free to diffuse through a membrane dissociates in the compartment where such dissociation is favored. This ionization reduces the concentration of the diffusible chemical so that a concentration differential is established. More of the diffusible chemical then migrates to the compartment where it can be dissociated more favorably until equilibrium between the two compartments with respect to this chemical is attained. The Donnan distribution effect may be a significant factor governing the flow of a weak acid from the stomach (pH 2 to 4) or the small intestine (pH 4.5 to 6.5) to the blood (pH 7.35 to 7.45). The degree to which the Donnan effect enhances the net transfer of a substance across the GI mucosa is highly dependent on the pH differential between the intestinal medium and the blood and the pKa of the substance. An example of this type of distribution is shown by the study of Shore and associates (1957), who injected acidic and basic drugs intravenously into dogs. In the gastric juice, where their ionization is favored, the concentration of basic drugs at equilibrium increased to as much as 40 times that in the plasma. In contrast, acidic drugs, whose ionization is not favored, either were absent or increased in concentrations to no more than six-tenths of those in the plasma. Zawoiski and colleagues (1958) also observed that the concentration in the gastric juice of an organic base injected into the dog increased as the pH decreased. The Donnan distribution effect is apparently less effective in the transfer of weak bases, such as some alkaloids, from the GI tract to the blood. This is because ionization in the blood is less favorable and, hence, there is a comparatively small, if any, concentration differential. The Donnan effect also occurs if one of the charged components in one compartment is a macromolecule too large to diffuse through the membrane. When this molecule dissociates, and one of the particles so formed is freely diffusible, the movement of this particle to the opposite side must be accompanied by a movement in the opposite direction of a particle of the same charge to maintain electrical neutrality. Thus, this process may cause the transfer of a different substance to the compartment containing the macromolecule. If, on the other hand, the compartment opposite that containing the macromolecule has freely diffusible positive and negative ions and the ions are the same as those associated with the macromolecule, then at equilibrium a net increase of these ions is found in the macromolecule compartment. This type of transfer of

V = 8πr4Phl where V = rate of flow r = radius of pore P = hydrostatic pressure or osmotic pressure h = viscosity of solution flowing through pore l = length of pore The extent of hydrodynamic flow across the membrane of intestinal epithelium differs from what can result from the osmotic gradients across the membrane of the small intestines. This difference is explained on the basis of the concept of bulk flow (Concon, 1988). The bulk flow can increase transport of solutes through the pore by “dragging” the molecules in the moving stream. The dragging effect may be expected to be greater at the time when absorption by the various mechanisms is at full capacity, so much so that the total osmolar concentration in the plasma is much greater than that in the intestinal lumen. In this case, the greater the total rate of absorption, the higher the total osmolar concentration of the plasma. Consequently, the dragging effect is also greater. The dragging effect will obviously be expected to be greatly increased when the amount of fluid ingested is large, since, in addition to osmotic effects, the hydrostatic pressure is also greater. However, because of the very nature of dragging and because of the relatively large molecules of many toxicants the contribution of the dragging phenomenon to the total absorption of toxicants is probably much less than that from other mechanisms, such as the active transport processes.


charged particles, to a large extent, may not be applicable in the case of the GI tract because charged particles are not freely diffusible across the GI membrane. Thus, it is essential that the charged substances that are important to life are transported by special mechanisms. A net transfer of a substance can also result from the Donnan distribution effect, if in one compartment, a nondiffusible substance, such as a protein, can bind the diffusible molecule. The movement of the diffusible substance bound by the protein follows Fick’s law, since in effect, a large concentration gradient is present as long as the binding capacity of the protein is not exhausted. 2.4.2 Special Transport There are a number of instances in which the movement of a compound across a membrane cannot be explained by simple diffusion or filtration because the compound is too water-soluble to dissolve in the cell membranes and too large to flow through the channels. Thus, if the GI absorption were to rely solely on passive transport, the limitations imposed on this process would exclude many compounds, even those essential to the survival of the organism. Those compounds that meet the conditions necessary for passive absorption in many cases cannot be absorbed at a rate commensurate with the needs of the body. Furthermore, the requirement of these substances by the body may be such that their concentration in the lumen of the GI tract may be lower than that in the blood. Therefore, even though the GI epithelium is an “open” membrane, absorption under these conditions is thermodynamically impossible. Instead of absorption, leakage into the GI lumen results. Thus, for the transport of many essential substances, such as sugars, amino acids, and nucleic acids, as well as some foreign molecules, the GI epithelium is equipped with a number of special transport systems. The special transport mechanisms thus are most manifest in GI absorption. These mechanisms attain much greater importance in the elimination of toxicants, however, in which special transport is important to the removal of xenobiotics and their metabolites. An important characteristic of the special transport systems, when operable, is that they allow movement of compounds with lesser lipid solubility, compounds that would ordinarily be expected to move very slowly through the highly lipophilic cell membranes. These processes are briefly described in the following. Active Transport Active transport requires energy and permits the absorption of the compound even against a concentration gradient. The following features characterize an active transport process:


1. 2.

3.

4.

Chemicals are moved against electrochemical gradients. The transport system is saturated and a transport mechanism exhibited at high substrate concentrations. The transport system is selective. Therefore, certain basic structural requirements exist for chemicals to be transported by the same mechanism with the potential for competitive inhibition. The system requires the expenditure of energy so that metabolic inhibitors block the transport process.

Compounds that are actively transported across a cell membrane are presumed to pass into the cell interior by forming a complex with a macromolecular carrier, generally proteins, on one side of the membrane. The complex subsequently traverses to the other side of the membrane, where the compound is released. The carrier molecule then returns again to the original surface to repeat the transport cycle. Carrier proteins involved in the active transport processes generally have specificities for certain kinds of chemical groups and configurations (Crane, 1979). This type of active transport system can be inhibited by a variety of compounds. For example, the transport of sugars and amino acids can be inhibited by cyanide, dinitrophenol, malonate, fluoroacetate, arsenate, and copper (Ther and Winne, 1971). The carrier system can also be saturated. This mechanism imposes a limit on the rate of absorption, even though the amount absorbed generally is a function of the dose. Many exogenous compounds can also compete with the carrier systems of essential nutrients, especially substances that structurally resemble the nutrients and other endogenous physiologically essential compounds. The active transport of organic chemicals appears to be closely associated with the sodium transport. Thus, compounds that inhibit sodium transport also inhibit the transport of the organic compounds (Ther and Winne, 1971; Concon, 1988). The energy-requiring active transport mechanism can also be inhibited by interference in the metabolic sources of energy. Thus, compounds that are inhibitors of oxidative phosphorylation also inhibit active transport of a variety of nutrients. Various sections of the GI tract seem to have specific preference for the active absorption of a compound or groups of compounds. For example, sugars as well as the neutral amino acids are largely absorbed in the middle portions of the small intestine, whereas the basic amino acids are absorbed equally in all parts of the small intestine (Concon, 1988). Bile salts and vitamin B12 are absorbed

mostly in the ileum; Ca 2+, Fe2+, and Cl– are absorbed mostly in the upper small intestine. In contrast, Na+ appears to be equally absorbed in all parts of the small intestine and colon; H + is absorbed most in the ileum and colon. The active transport process is of fundamental importance in toxicology. It is involved in the elimination of foreign compounds from the organism. To transport substances out of the cerebrospinal fluid, the central nervous system has two transport systems at the choroid plexus, one each for organic acids and organic bases (Klaassen, 1986a). Likewise, the kidney has two active transport systems that eliminate foreign compounds from the body, and the liver has at least four active transport systems, two for organic acids, one for organic bases, and one for neutral organic compounds. The active transport system itself is also potentially a target of many toxic compounds, which inhibit the process in one way or another. From the nutritional standpoint, active transport’s being subject to competitive inhibition even among the nutrients is also relevant to the toxic effects of nutrient excesses.

cess therefore explains the absorption of protein toxins and other toxic materials that otherwise would be excluded from the intestinal epithelium for reasons of molecular size alone. Since many toxic substances in foods are macromolecules, their toxicity is therefore related to the ability of the small intestine to absorb them.

2.5

FACTORS AFFECTING INTESTINAL ABSORPTION

The toxicity of compounds absorbed through the GI tract is generally much less than that of compounds that gain entry through other routes. This is because the GI tract imposes certain limitations on their rates of absorption. Most substances absorbed in the GI tract must pass through the liver, where they can be metabolized to derivatives of lesser or greater toxicity. In addition, several other factors can influence the absorption of toxicant through the GI tract. Their importance in the manifestations of toxic effects is briefly described. 2.5.1 Effect of Blood Flow

Facilitated Transport The facilitated transport mechanism is similar to active transport except that in this carrier-mediated transport system, the substrate is not moved against a concentration gradient. Also, this process does not require any energy expenditure. Hence, the term facilitated diffusion was coined by Danielli (1954). The transport of glucose from the GI tract into blood, from plasma into red blood cells, and from blood into the central nervous system is thought to occur through facilitated diffusion. There is evidence that facilitated transport may also apply to exogenous chemicals (Fiese and Perrin 1969). Similarly to active transport, facilitated diffusion is also subject to saturation phenomena, competitive inhibition of similar compounds, stimulation by sodium ions, and a temperature effect. Endocytosis Pinocytosis (for liquids) and phagocytosis (for solids) are specialized transport processes in which the membrane invaginates or flows around a toxicant, allowing more ready transfer across cell membranes. This type of transfer across cell membranes is important to removal of particulate matter from the alveoli by alveolar phagocytes and for removal of some toxic substances from the blood by the reticuloendothelial system of liver and spleen. A defect in the intestinal epithelium may enhance pinocytosis of macromolecules (Concon, 1988). This pro-


Compounds that influence the blood flow generally also influence the rate of absorption. Thus, vasoconstrictive drugs such as serotonin, norepinephrine, and vasopressin diminish blood flow and consequently absorption of water. In contrast, ethanol, which increases the blood flow rate, is absorbed at a rapid rate in the stomach (Magnussen, 1968). Blood flow can also influence absorption by its effect on the supply of oxygen and other nutrients. This property follows from the fact that active transport requires oxygen. Indeed, there appears to be a critical blood flow rate through the splanchnic area below which active transport ceases (Robinson et al., 1964, 1966). The loss of active transport capacity of the intestinal epithelium is also observed in intestinal ischemia. Such loss of active transport capacity can be prevented by perfusion of the ischemic tissues with solution saturated with oxygen. The draining effect of blood increases the rate of absorption simply from a consideration of Fick’s law, since the removal of absorbed substances in the serosal side of the GI epithelium maintains a large concentration gradient. Normally, the rate of blood flow in the portal vein (Figure 2.2) is approximately 1.2 L/hr/kg, with a 30% increase in blood flow through the splanchnic area after a meal (Concon, 1988). Therefore, an increased absorption of toxicant may result if it is ingested during a meal, assuming that the pH is favorable and the toxicant is not bound to other components in the food. As a corollary, a decrease in blood flow rate lowers the intestinal absorption

of toxicants. Thus, a normal blood flow rate in the intestinal epithelium is essential to maintenance of a normal rate of absorption of chemical; compounds that increase the blood flow generally displaying an enhanced toxicity. 2.5.2 Effect of Lymph Flow Rate The lymphatic flow rate, only about one-six hundredth to one-thousandth of that of the blood, is important for highly toxic substances, such as the botulinum toxin, that are transported by the drag effect and bulk flow mechanism. However, very little is known about the effect of lymph flow rate on the absorption of exogenous toxicants. 2.5.3 Gut Motility and Emptying Time Gut motility and the rate of passage and elimination of food from the GI tract also influence the rate of absorption of chemicals. Higher absorption rates are observed with increased residence times of food in the GI tract. Therefore, any conditions that decrease or increase gut motility and emptying or passage time have a corresponding effect on the toxicity of compounds. If smaller amounts of compounds are presented slowly for absorption in the GI tract, there may be sufficient time and several ways to dispose of the undesirable compounds. Consequently, a toxicologically insignificant amount, if any, ultimately reaches its target organ or tissue. The emptying or passage time in the GI tract can also influence the toxicity of compounds. The intestinal microflora play an important role in this regard. For example, a compound that is made toxic by action of the intestinal microflora may show no toxic effects if it moves slowly through the intestinal tract. The reason is that only small amounts unabsorbed by the intestine reach the colon, where microflora are most abundant. In contrast, a substance may move rapidly in the GI tract for various reasons (e.g., stimulation by cathartics), so that a fraction of the toxic dose is absorbed. The same reasoning applies to stomach emptying for those compounds absorbed in the ileum or colon. The rate of appearance of substances in the colon also regulates their metabolism by the microflora, influencing both their absorption and their toxicity. The gastric emptying time is influenced by the type and volume of the meal, the acidity of the gastric content, the neutralization process in the duodenum, and drugs such as the pressor amines, norepinephrine, histamine, and tyramine (Levine and Walsh, 1975; Holz, 1968; Concon, 1988). These drugs are found in many types of food products even though their concentrations are often too low to affect normal intestinal function. They also have potent ef-


fects on the motility of the intestines and colon (Holz, 1968). Microbial infections of the intestinal wall and other disease symptoms can also affect the intestinal motility. Such diseases produce a rapid transit time of intestinal contents, as in diarrhea, or the opposite effect, as in constipation. These conditions are extreme examples of contrasting effects of gut motility and transit time. Pathological conditions in the GI tract may also affect the integrity of intestinal mucosa and the toxicant absorption. For example, because many toxic substances are lipid soluble and are absorbed and transported in association with lipids via the lymphatic system, any malabsorption of fats may have far-reaching toxicological implications other than those involving these substances directly. 2.5.4 Chemical Factors Affecting Absorption Chemicals may affect the absorption of compounds by (a) formation of insoluble precipitates, or complexes with specific substances, or formation of chelates that facilitate or inhibit absorption and solubilization; (b) competition for binding or carrier proteins involved in absorption; and (c) modification of the motility or absorptive capacity of the GI mucosa. Compounds such as phytic acid, oxalates, and gossypol can form insoluble complexes with bivalent metal ions, amino acids, and proteins. Insoluble precipitates can also be formed by phosphates, fatty acids, and alkalis (e.g., antacids). Sometimes, chelation can also improve the absorption of toxicants. For example, citric acid can increase the absorption of lead (Graber and Wei, 1974), and magnesium improves the absorption of dicoumarol (Ambre and Fischer, 1973). Competition for carrier proteins also influences the absorption of toxicants. In this regard, similarity in general structure may be sufficient to influence the absorption of compounds. Toxicants can also modify the absorptive capacity of the intestinal mucosa by interaction with its structural constituents. Lectins, for example, bind strongly with specific receptors in cell membranes. The absorptive capacity of the intestinal mucosa may be affected by changes in the acidity of the intestinal mucosa. Thus, compounds that inhibit carbonic anhydrase cause a decrease in intestinal pH (Concon, 1988). The general metabolic integrity of the GI mucosal tissues is thus essential to their structural and functional status. Any substance or condition that destroys the metabolic integrity of these tissues has an adverse effect on

their function and structure, thereby affecting the absorption of compounds.

The specific classes of molecules and their potential sensitivity to alterations by toxicants are briefly described in the following sections:

2.6

Proteins

BIOLOGICAL TARGETS OF TOXIC COMPOUNDS

A knowledge of the interactions of a toxicant with specific molecular targets, the role that molecular target plays in the chemical dynamics of the cell, and the response of the cell to either the presence of the toxicant or the perturbations the toxicant elicits is fundamental to our understanding of the manifestations of toxic effects. In this section, the potential molecular, subcellular, and cellular targets available to a toxicant for interaction are briefly described. 2.6.1 Molecular Targets The four basic macromolecules, viz., proteins, lipids, carbohydrates, and nucleic acids, involved in the dynamic execution of living processes in the biological systems are frequently the target of toxic compounds. The small metabolites of the cell are quickly and easily replaced after modifications, since they are part of the flux of material throughout the metabolic pathways. In contrast, the macromolecules are of complex biosynthetic origin. Their replacement in the cellular system is energy intensive and requires a dietary supply of precursors, such as essential amino acids, fatty acids, and vitamins. Generally, interactions of toxicant with macromolecules, especially cellular enzymes involved in important metabolic pathways, often result in improper levels (excessive or deficient) of a cellular component. This effect, in turn, may produce a range of subtle but pervasive effects, varying from a disturbance of the osmotic strength of the cell’s cytoplasm to the interruption of energy metabolism. In the cell, one perturbation may trigger another, in a cascading series of reactions that may intensify the potential for harm and, spatially and temporally, obscure the initial triggering reaction. At some point in the series of reactions, the system is irreversibly altered, even dies. At present, our knowledge of such interactions at cellular level is confined to those of a relatively few wellcharacterized toxicants, especially those that are very specific in action and are potent, and in which the exposure produces an acute effect. The toxicity of compounds that either act chronically by a mechanism different from that of their acute action or produce latent symptoms (e.g., the mutagens, carcinogens, and teratogens) is extremely difficult to describe.


Proteins are the first product of a cell’s genetic potential. They can be broadly grouped into the following five categories: 1. 2. 3. 4. 5.

Structural proteins, e.g., collagen Catalytic proteins, e.g., various enzymes Carrier or storage proteins, e.g., hemoglobin, transferrin, ceruloplasmin Informational or regulatory proteins, e.g., peptide hormones such as insulin or repressor proteins Immunological proteins, e.g., immunoglobulins involved in defense mechanisms

Specific proteins that may fill several roles simultaneously are not unusual. Toxicants primarily interact with the side chains of amino acids that constitute the primary backbone of the proteins. Because these side chains are also involved in and primarily determine the secondary and tertiary structures of the protein, their interactions with toxicants can disturb protein structure. Usually, a disturbance at any level of protein structure, especially enzymes, can alter the protein’s catalytic or biological function. The relative order of nucleophilicity (i.e., capacity of any atom containing an unshared pair of electrons or an excess of electrons to participate in covalent bond formation) relative to the major groups in biological molecules can be summarized as follows: R-S– > R-SH R-NH2 > R-NH3+ R-COO– > R-COOH R-O– > R-OH R-OH = H-OH and finally R-S– > R-NH2 > R-COO– = R-O– Amino acids whose side chains are capable of reacting with toxic chemicals are listed in Table 2.3. From the preceding relationships it is obvious that the strongest nucleophile in protein molecules is the sulfhydryl group of cysteine, particularly in the ionized, thiolate form. Next in line are the amine groups in their uncharged, unprotonated forms, including the α-amines at the N terminals, the ε-amines of lysyl side chains, the secondary amines of histidine imidazolyl groups and tryptophan indole rings, and

Table 2.3 Amino Acids with Side Chain Functionalities and Their pKa Values Amino acid All All Lysine Histidine Arginine Tyrosine Aspartic acid Glutamic acid Cysteine

Functional group

pKa range

α-Amino, N terminus α-Carboxyl, C terminus ε-Amino Imidazolyl nitrogen Guanidinyl group Phenolic hydroxyl β-Carboxyl γ-Carboxyl Sulfhydryl

7.6–8.0 2.1–2.4 9.3–9.5 6.7–7.1 > 12 9.7–10.1 3.7–4.0 4.2–4.5 8.8–9.1

the guanidino amines of arginine residues. Finally, the least potent nucleophiles are the oxygen-containing ionizable groups, including the α-carboxylate at the C terminals, the β-carboxyl of aspartic acid, the γ-carboxyl of glutamic acid, and the phenolate of tyrosine residues. These side chains of amino acids in a protein molecule are predominantly involved in interactions with various toxicants. Coenzymes Coenzymes are a class of biomolecules that participate in several enzymatic reactions and are present in limited concentrations within the cell. Their synthesis is complex, and their replacement is in part dependent on the dietary supply of vitamins. The coenzymes themselves may be subject to direct attack by toxicants. For example, some symptoms of heavy metal poisoning are similar to those of vitamin B1 deficiency. Metalloproteins, especially the heme proteins possessing the iron-containing protoporphyrin ring, are also quite vulnerable to attack. Cyanide poisoning of cytochrome c oxidase, a heme-containing protein involved in the terminal electron transport process during respiration, is well known. Nucleic Acids Nucleic acids are the building blocks of a cell’s genetic material, viz., the deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). The DNA molecule offers a target of considerable size for interaction with toxic chemicals. The introduction of an error in the DNA results in the loss of quality or quantity of biological information. The error produces a faulty protein molecule or results in a level of an RNA and/or protein species that is inappropriate to the cell’s state of differentiation. A permanent modification of DNA is a mutation, which, if expressed, may lead to a carcinogenic and teratogenic event.


Aside from the inhibition of enzymes involved in their synthesis, toxic compounds may also affect DNA and RNA formation and function by reacting with the macromolecules themselves. For example, DNA replication, RNA translation, and consequently protein synthesis may be interfered with by alkylation of the DNA or RNA purine or pyrimidine bases by N-nitroso compounds, such as the nitrosamines. Lipids Lipids primarily serve three cellular functions: storage, structural, and informational. Triglyceride stores in the cell and the adipose tissue are mobilized in times of stress or food deprivation to yield fatty acids and glycerol for energy production. This process is not regarded as an essential function. However, lipids are an integral part of membranes. Their length, their degree of unsaturation, and the nonlipid moieties attached to them essentially govern the permeability, excitability, and fusion properties of the membrane, as well as influence the activity of membranebound enzymes complexes. Lipids, in the form of steroids, also serve as hormones. The most susceptible function to interactions with exogenous toxicants seems to be related to the lipid’s role in membrane structure and function. Free radicals generated by exogenous agents can cause oxidative changes in the unsaturated fatty acid constituents of the membranes. Such oxidative changes may in turn lead to carcinogenesis, mutagenesis, and cellular aging mechanisms. Disturbances in steroid metabolism may also lead to cancer. Carbohydrates Carbohydrate polymers serve structural, recognition, and storage functions in the cell. Carbohydrate moieties on the surface of a cell are also involved in regulation by the body’s immune system. Cells that have been transformed to tumor cells display an altered carbohydrate surface determinant. Generally, any substantial modification of carbohydrate structure or function occurs by modification of the enzymes involved in carbohydrate metabolism. The molecular weight and the redundancy of their structure in the cell simply render them too diffuse and inert to suffer attack directly by an exogenous chemical. Thus, as compared to other macromolecules, carbohydrates are not sensitive or frequent targets of exogenous chemicals. 2.6.2 Subcellular Targets Many toxicants show little discrimination in their attack on molecular targets in vitro but do elicit specific patho-

logical effects in vivo. Thus the accessibility of a particular cellular structural component, as well as the component’s influence on the integrity of one or another organelle, determine the specificity of attack. Organelles possess enzyme systems specific to their purpose. This compartmentalization and specialization of function explain in part the susceptibility of one organelle to a toxicant to which another is impervious. The availability of such targets also explains why a toxicant elicits one type of damage in one cell population but has a totally different effect in another. Some specific subcellular targets available for the exogenous toxicants are described next.

compounds makes this subcellular system a frequent target of toxicants. The activity is particularly high in the liver, although the system exists to a smaller extent in most cells. In some instances, the products formed by the enzyme system of the ER are more toxic to the cell than the parent compound. Furthermore, since the enzymes of the ER metabolize primarily endogenous substrates, unnatural proliferation of this system may result in abnormal levels of hormones, bile salts, and other normal metabolites. Thus an excess of enzyme activity can be as dangerous to the biological system as a deficiency.

Nuclei

The plasma membrane that surrounds the cell has considerable microheterogeneity in its structure and function. It is home to, among others, the cell antigen-recognition sites, membrane transport enzymes such as the Na+-K+ pump, and hormone receptor sites. Given the natural messengers and activators that arrive and operate at this dynamic interface, it is not surprising that several toxins also interact with and attack the plasma membrane. In fact, a general feature of cellular injury seems to be an increase in the permeability of the plasma membrane. This results in an influx of sodium and calcium ions and an efflux of potassium ions. A loss of soluble proteins from the cell may finally occur. Although it is not possible to decide whether to attribute the change in permeability to a primary attack of the toxicant on the membrane directly, a loss of selective permeability by the plasma membrane is unquestionably one of the characteristics of a dying cell.

Nuclei are the site of DNA and RNA synthesis. Toxicants can interfere with the synthesis of both DNA and RNA, generally by altering the activity of the individual enzymes involved. Mitochondria The mitochondria of the cell are the sites of oxidative phosphorylation and, therefore, are primarily responsible for adenosinetriphosphate (ATP) synthesis. A number of toxicants, some ancient and notorious, attack various parts of the mitochondrial oxidation system, producing low ATP levels and disturbance of the redox state of the cell. Lysosomes Lysosomes are intracellular vesicles of hydrolytic enzymes, including nucleases, phosphatases, and peptidases. The vesicle normally sequesters these potent hydrolases from the cytoplasm. A disruption of the lysosomal membranes by interaction with toxicant releases the hydrolytic enzymes to attack adjacent cell material.

Plasma Membrane

2.6.3 Cellular Targets The susceptibility of a cell to a toxin primarily depends on at least the following three factors: 1.

Endoplasmic Reticulum The endoplasmic reticulum (ER) is the fine filigree of intracellular membrane sheets that, upon cell disruption, yield the microsomes. The ER is divided into two basic units, rough and smooth, the former masked by the attachment of ribosomes. The ER also contains electron transfer enzymes responsible for oxidation of various lipophilic compounds, including the steroids, long-chain fatty acids, and exogenous compounds. Perturbation of the ER upon exposure to and interaction with a toxicant may result in a disturbance of the membrane and constituent enzyme activities, or a proliferation of the ER structure and/or specific enzymes attached to it. The ability of the ER to metabolize foreign


2. 3.

The specialization of the cell, i.e., which susceptible organelles are preeminent in the cell’s economy The distribution of the toxin within the body The cell’s reaction to the presence of the toxin

An example of the first type, i.e., selective toxicity based on cell specialization, is the sensitivity of the cells of the myocardium to anoxia. These cells depend primarily on ATP generated by mitochondrial oxidative processes and, hence, are critically aerobic. An interruption of the blood supply (ischemia) quickly produces cell death (an infarction). Another example of selective toxicity based on specialization is the destruction of rapidly dividing intestinal crypt cells by DNA synthesis inhibitors, such as the nitrogen mustards.

The distribution of a toxin in the body and its route of entry and elimination are also important factors. Since a toxin rarely has homogeneous distribution, it is inevitable that some cell populations suffer high exposure. For example, the red blood cells, because of their thorough exposure to dissolved gases in the lungs and their high level of hemoglobin, are primary targets of carbon monoxide poisoning. The liver, in contrast, is infused with the blood directly from the stomach and small intestine. The nutrients, as well as all ingested toxins, therefore, impact initially on this organ. Finally, what the cell can or cannot do with the toxin also determines its relative sensitivity to exogenous toxicants. The liver has a large complement of enzymes in the microsomes that can metabolize a wide range of exogenous chemicals. In many cases, the liver is successful in eliminating or decreasing toxicity, but in some instances, it may create a more toxic metabolite to its own detriment. Other cells, with less active microsomal enzymes, either are less effective in dealing with a toxicant or are more resistant to it, depending on whether metabolism deactivates or activates that compound. It should also be emphasized here that the cell is remarkably resistant and can survive temporary disturbances

Figure 2.6

in its environment. Similarly, during the course of evolution, it has also developed several defensive mechanisms to counterattack the effects of exogenous chemicals in biological systems. Some of these mechanisms are described in Chapter 5. They are the emergency measures that operate with varying degrees of success and efficiency.

2.7

BIOCHEMICAL EFFECTS RESULTING IN TOXIC INJURY

Because the cell’s components are reactive chemicals, toxicants can react with its components and interfere in its operation. Exogenous substances that interfere in the cell’s activity are called intrinsic toxicants. Those native or familiar to the cell and toxic only when present in excess are called relative toxicants (Concon, 1988). Many of the compounds of the latter group are in fact essential to the normal operation of the cell; others are metabolic byproducts. As shown in Figure 2.6, a toxicant may be detoxified by metabolic processes and eliminated from the body, made more toxic (toxified) by metabolic processes and distributed to receptors, or passed on to receptors as a met-

Major steps involved in the overall process leading to toxic effects of chemicals.


abolically unmodified toxicant. In every biological system, there are thus safeguards against the intrusion of unwanted chemicals. However, these safeguards are imperfect. When they fail, toxic effects result. As discussed in the preceding section, toxic effects eventually indicated by gross functional disturbances must necessarily initiate injury at the molecular level. Although the biochemical mechanisms of toxicity of a large number of compounds are still unknown, the biochemical bases of their toxicities may include one or more of the following general types. 2.7.1 Deficiency of Essential Compounds Nutrients that cannot be synthesized by higher living forms necessarily must be supplied from external sources. The urgency of delivering these compounds to the cell depends on their rate of usage and degradation, the extent of cell storage, and the magnitude of their involvement in energy production, the cell’s primary need. Nutrient deficiencies in cell can occur either through the absence of nutrients in foods, a primary deficiency, or failure of or interference in their delivery or metabolism. Such an internal obstruction is termed a secondary deficiency. One of the following mechanisms may be responsible for such failure. Inhibition of Digestive Enzymes or Other Digestive Factors Toxicants can inhibit a variety of enzymes involved in the digestive processes. Well-known examples include inhibitors of the proteolytic and amylolytic enzymes present in several legumes. Absence of Digestive Enzymes or Other Digestive Factors Lactose intolerance in certain segments of populations is well known because of the inherent genetic deficiency of β-galactosidase enzyme. Similarly, certain oligosaccharides present in legumes cannot be metabolized because of the absence of corresponding digestive enzymes. Interference in the Absorption of Essential Compounds Interference in the absorption of essential compounds may arise as a result of the following factors: 1.

2.

Chemical or physical combination of one compound with another, resulting in the formation of a nonabsorbable complex: chelation of dietary essential minerals by phytate falls in this category. Absence of the compound necessary for the absorption of the compound: for example, vitamin


3.

4.

5.

6.

B12 is not effectively absorbed in the absence of the intrinsic factor. Interaction or modification of the GI mucosa: An example is the group of compounds known as lectins, which bind on the absorptive surfaces of the intestinal mucosa. Inhibition of enzymes involved in absorption: for example, the antibiotic actinomycin D inhibits RNA and protein synthesis, including presumably enzymes necessary for the absorption of amino acids (Yamada et al., 1967; Concon, 1988). Solubilization of essential compounds in solvents that are nonabsorbable. For example, mineral oil, which may dissolve lipid-soluble vitamins, may prevent their absorption. Increased motility of the GI tract: many factors can affect this motility. Certain factors in foodstuffs may cause diarrhea and similar rapid evacuation of intestinal contents, resulting in poor absorption of essential compounds.

Interference of the Transport of Essential Compounds to the Cells Nitrite ions, for example, interfere in the transport of oxygen to hemoglobin. Degradation of Essential Compounds Nutrients may be destroyed even before they are absorbed. For example, there are factors, such as thiaminases, that destroy thiamine; retinol and carotenoids may be destroyed by oxidizing agents; and ascorbic acid, by ascorbic acid oxidase. Inactivation of Essential Compounds Certain substances may react with some of the essential nutrients without causing degradation but rendering them biologically inactive. Cyanide, for example, may interact with cobalamin, vitamin B12, to form cyanocobalamin, which is biologically inactive. It is believed that chronic cyanide intoxication may be the cause of tropical amblyopia as a result of cyanide inactivation of cobalamin. Interference of the Uptake of Essential Compounds in the Cells or Tissues Thiocyanate inhibits the iodine uptake of thyroid cells, whereas glucose and amino acid uptake of muscle cells does not occur in the absence of insulin (Concon, 1988; Langer and Stolc, 1964).

Antagonism Between Essential Compounds Antagonism exists among leucine, isoleucine, and valine when one of these is present in relatively large excess over another (Harper et al., 1970); β-carotene and cholecalciferol are also antagonistic (Weits, 1964). 2.7.2 Inhibition of Metabolic and Other Nondigestive Enzymes Several toxicants inhibit the activity of metabolic and other nondigestive enzymes. Carbamates and organophosphate pesticides, for example, are potent inhibitors of acetylcholine esterase, an enzyme necessary for neurotransmission (Aldridge and Reiner, 1969; Concon, 1988, O’Brien, 1969a, 1969b). The inhibition of monoamine oxidase by antidepressant drugs has been shown to increase the biological activity of pressor amines found in several foods (Vettorazzi, 1974). 2.7.3 Interference with Neurotransmission Several aspects of the neurotransmission process are susceptible to the action of various toxicants. The propagation of the nervous impulse is rapid and is obviously an energyrequiring process. The complexity and rapidity of neurotransmission may also lay bases for its vulnerability. The interference of several toxicants with this process is usually quite serious and often fatal. For example, the deadly poisons tetrodotoxins, from the puffer fish, and saxitoxin, from shellfish and clams, derive their lethality from their capacity to block specifically the sodium gates of the axon (Kao, 1972; Narahashi, 1972). In contrast, DDT acts in an opposite way, by keeping the sodium channel open but partially blocking the potassium channel (Narahashi and Haas, 1967). The synaptic mechanism by which the nerve impulse propagates appears to be the specific target of many potent toxicants. The release of the neurotransmitter acetylcholine can be blocked by botulinum toxin (Brooks, 1956). Acetylcholine esterase, involved in the regeneration of acetylcholine after the transmittal of the nervous impulse, is also a target of many poisons, such as organophosphates (O’Brien, 1969a, 1969b) and the cholinesterase inhibitors in the potato, eggplant, tomato, and sugar beet (Orgell, 1963). Many exogenous substances can also mimic the effects of neurotransmitters. Muscarine, the toxic alkaloid from the mushroom Amanita muscaria, behaves in the same way as acetylcholine (Bradley et al., 1966). Its extreme toxicity is attributed to its resistance to degradation in the tissues.


Toxic reactions directly involving the nervous system are generally more severe, with an almost immediate appearance of symptoms. Furthermore, other toxicity mechanisms, such as interference in the transport of nutrients, protein synthesis, energy metabolism, and respiration, indirectly affect the nervous system. 2.7.4 Phototoxic Reactions Certain exogenous and endogenous compounds in the skin cells, when sufficiently illuminated, may become highly reactive with cellular components. This process by which light damages tissues, in the presence of a photosensitive substance, is called phototoxic reaction and is also known as photosensitization or photodynamic action. Ippen (1969) classified two types of phototoxic reactions: photoautoreaction and photoheteroreaction. In the first type, the photosensitive substance merely induces the normal photochemical reaction of the cell as in sunburn formation. In other words, in the presence of the phototoxic compound, skin may become more readily susceptible to the deleterious effects of sunlight. Most phototoxic reactions, such as those caused by furocoumarins, are of this type. In the second type, a toxic product is formed from the photochemical reaction of the photoactive substance. This toxic derivative may be formed independently of the tissues. Chlorpromazine and sulfanilamide are examples of compounds that are involved in phototoxic reactions of the second type. Photoallergic reaction is a form of photoheteroreaction. In this case, the photochemical reaction produces an allergen. The classes of phototoxic compounds include both natural and synthetic compounds. The natural compounds include the hypericins (Blum, 1964), the furocoumarins or psoralens (Pathak, 1969), porphyrins (Clare, 1956; Rimington et al., 1967), steroids, essential oils (Spikes, 1968), riboflavin (Spikes and Glad, 1964), and flavin mononucleotide (FMN) (Frisell et al., 1959). The synthetic phototoxic compounds include many drugs prescribed routinely, such as anesthetics, antibiotics, antihistamines, diuretics, barbiturates, sulfonamides, phenothiazines, dyes and other coal tar and petroleum products, and perfumes and colognes (Spikes, 1968). 2.7.5 Interference with Genetic Material and Function Interactions of toxicants with DNA and RNA, the building blocks of a cell’s genetic material, not only affect cellular reactions such as protein synthesis, but may also lead to mutagenesis and carcinogenesis. These processes are described in greater detail in Chapter 4. Mutagenic effects in-

volving the germinal cells have the potential for hereditary transmission. Therefore, the damage of these effects can extend beyond one generation and across pedigrees and population groups.

2.8

DISTRIBUTION OF TOXICANTS

Subsequently to absorption, the toxicant is capable of distribution (translocation) throughout the biological system. The transport processes discussed earlier are the major factors operative in the distribution of the toxicant from cell to cell and organ to organ and for movement into total body water. Body fluids are distributed among three primary components: plasma water, interstitial water, and intracellular water. Vascular fluid has the important role in the distribution of absorbed toxicants. Human plasma accounts for approximately 4% of body weight but 53% of total blood volume, whereas the interstitial tissue fluids account for 13% of body weight and intracellular fluids for 41% (Guthrie and Hodgson, 1987b; Klaassen, 1986b). Although only a small amount of toxicant in the body may be in contact with the receptor or target site, it is the distribution of the bulk of the toxicant that governs the concentration and disposition of that critical proportion. If the toxicant is distributed only in plasma, a high concentration is achieved in the vascular tissue. On the contrary, if the same quantity of toxicant is also distributed in the interstitial and intracellular water, concentrations are much lower in the vascular system. The rate of distribution of the toxicant to the tissues of each organ is thus primarily determined by the blood flow through the organ and the ease with which the chemical crosses the capillary bed and penetrates the cells of the particular tissue. Its eventual disposition is largely dependent on the ability of the toxic chemical to pass through the cell membranes and its affinity for the various tissues. The following are some of the factors that affect the distribution of the toxicant in biological systems. 2.8.1 Binding to Plasma Proteins Binding by the plasma proteins has an important bearing upon the distribution of toxicants. Serum albumin is the most important protein in this regard. Because many toxicants are very lipophilic, the plasma lipoproteins also play an important role in toxicant binding. The binding is noncovalent, involving hydrogen, Van der Waals, and ionic bonds, and the proportion of the toxicant bound depends on various physicochemical factors.


The nonbound (free) portion of the toxicant in the plasma is in equilibrium with the bound portion, but only the former passes through capillary membranes. Therefore, excessively protein-bound compounds (>90%) are restricted in terms of equilibrium (distribution) within the organism. Under steady-state conditions, the concentration in the extravascular water equilibrates with the free concentration in the plasma. Plasma protein binding sites may be saturated, or one bound compound may be displaced by another. Thus, a dose threshold for toxicity is often seen as a result of saturation of plasma protein binding sites, which results in a dramatic increase in the plasma concentration of the free compound. Such competitive binding for the same sites on a protein can have an important toxicological significance. This is especially true for highly toxic compounds possessing a very high affinity for protein binding sites. Although extensive plasma protein binding affects passive diffusion, which is concentration dependent, it has little effect on active transport processes such as secretion at the kidney. Plasma protein binding of toxicants therefore influences the distribution and the half-life of the toxicant in the body and is responsible for toxic dose thresholds. A number of methods are used to study toxicant(ligand)-protein interactions, including ultrafiltration, electrophoresis, equilibrium dialysis, solvent extraction, solvent partition, ultracentrifugation, spectrophotometry, and gel filtration or equilibrium. Such methods yield data that are often expressed in terms of the percentage of ligand bound. However, it must be noted that as ligand concentration is lowered, percentage of bound ligand increases. Thus, if a protein has a high affinity for a ligand, as often occurs with albumin, the percentage bound falls sharply when the total ligand concentration exceeds a critical value. 2.8.2 Plasma Level The plasma level of a toxicant is an important parameter in distribution as it relates more readily to the effect than the dose itself. In general, the plasma concentration more nearly reflects the concentration at the site of action, although the relationship may not be a simple one if the toxicant is sequestered in a particular tissue or organ. 2.8.3 Tissue Localization The passage of exogenous chemicals into cells and across membranes, as discussed earlier, is generally restricted to the nonionized, lipid-soluble form of the chemical. Thus, compounds that meet these criteria pass out of the blood,

diffuse through tissues, and are distributed through the body. Lipid-soluble compounds may dissolve in tissues with a high fat content and may remain sequestered there for some time. Some compounds may accumulate in a specific tissue because of their affinity for a particular macromolecule. Such is the case with the binding of carbon monoxide with hemoglobin. These sites may be the target sites for toxicity. Accumulation may also occur in tissues other than the site of action. Such storage depots for toxicants in the biological systems are described later in this chapter. Toxic compounds thus may be distributed throughout all the tissues of the body, or they may be restricted to certain tissues. Two areas for special consideration are the brain and the fetus. The blood-brain barrier does not completely prevent the passage of toxicants into the central nervous system (CNS) but rather represents a site that is less permeable than most other areas of the body. Therefore, many poisons do not enter the brain in appreciable quantities. The following are the major anatomical and physiological reasons why some toxicants have reduced capacity for entering the CNS (Klaassen, 1986b): 1.

2. 3.

The capillary endothelial cells of the CNS are tightly joined, leaving few or no pores between the cells. The capillaries of the CNS are largely surrounded by glial cell processes (astrocytes). The protein concentration in the interstitial fluid of the CNS is much lower than elsewhere in the body.

Thus, in contrast to other tissues, the toxicant has difficulty moving between capillaries and has to traverse not only the capillary endothelium itself, but also the membranes of the glial cells in order to gain access to the interstitial fluid. Furthermore, the low protein concentration of the interstitial fluid of the CNS also decreases the distribution of chemicals to the CNS. These features together act as a protective mechanism to decrease the distribution of toxicants to the CNS and thus the toxicity. In contrast, passage of compounds across the placenta occurs generally by passive diffusion. Lipid-soluble compounds are thus readily transported. However, if metabolism in utero converts the compound into a more polar metabolite, accumulation may occur in the fetus. Exogenous compounds generally achieve the same concentration in fetal plasma as in the maternal plasma water. In addition to chemicals, viruses (e.g., rubella, human immunodeficiency virus [HIV]), cellular pathogens (e.g., syphilis


spirochete), antibody globulins, and even erythrocytes traverse the placenta (Goldstein et al., 1974). 2.8.4 Volume of Distribution As mentioned earlier, body fluids are distributed among plasma and interstitial and intracellular water. The distribution of the toxicant into each of these three fluids profoundly affects the plasma concentration. If a toxicant is distributed only in the plasma water (approximately 3 liters in the average human), the plasma concentration is obviously much higher than if it is distributed in all extracellular fluid (approximately 14 liters) or the total body water (approximately 40 liters). The volume of distribution (VD) may be calculated from VD = dose (mg)/plasma concentration (mg/L) and is expressed in liters. A more rigorous determination of the volume of distribution utilizes the area under the plasma concentration/time curve (area under the curve [AUC]) (Figure 2.7) for the calculation: VD = dose/(k × area) where k is the elimination rate constant or VD = dose/C0 where C0 is the plasma concentration at time zero gained by extrapolation of the log plasma concentration versus time plot (Figure 2.8). Ideally, the compound should be administered intravenously, unless the degree of absorption is known. The parameter VD yields useful information. For instance, a very high apparent VD may indicate sequestration in a particular tissue or localization in fat. Similarly, the total amount of a toxicant in the body, i.e., the total body

Figure 2.7 Plasma level profile for a foreign chemical. AUC, area under the curve. (From Timbrell [1982]).

Figure 2.8 A semilog plot of the plasma level of a foreign chemical against time. C0, plasma concentration at time zero. (From Timbrell [1982].)

burden, may be estimated from a knowledge of the plasma concentration and VD:

A simple linear relationship is seen if the distribution of the compound fits a single compartment model, i.e., the toxicant being distributed in plasma water alone. If the toxicant first undergoes distribution and the plasma concentration declines more slowly, governed by the process of elimination and metabolism, then a two-phase decay is seen. For detailed mathematical treatment of the topic, the readers are referred to several excellent reviews (Goldstein et al., 1974; Tuey, 1980; Klaassen, 1986b; Gibaldi and Perrier, 1982). The half-life of a toxicant reflects the various processes taking place in vivo after the administration of a compound. Thus, following the initial absorptive phase, the toxicant is distributed, metabolized, and excreted, and these processes, acting in conjunction, determine the rate of removal of the toxicant from the plasma. Changes in the half-life of the toxicant may therefore yield valuable information about changes in these processes. For example, the half-life indicates the ability of the body to metabolize and excrete the compound. When this ability is impaired, either through saturation of enzymatic or active transport processes or if the liver or kidneys are damaged, the halflife may well be prolonged. An indication of the ability of the body to metabolize and eliminate the compound may be gained from the total body clearance. This may be calculated from the parameters described earlier.

Total body burden (mg) = plasma concentration (mg/L) × VD (L) Only the free rather than the total amount in the plasma should be used for the calculation of VD, as only the former is available for distribution. 2.8.5 Plasma Half-Life The plasma half-life is also an important parameter. It can be calculated from measurements of the plasma level at the various time points. The half-life is the time required for the plasma concentration of the toxicant to decrease by half from a given point. Measurement of the plasma level of a toxicant at various times after dosing gives a curve that decays exponentially as shown in Figure 2.7. Plotting the data semilogarithmically (Figure 2.8) gives a linear relationship from which the half-life can be readily calculated as follows: log C = log C0 – (kt/2.303) Slope = –k/2.303 Half-life (t1/2) = 0.693/k where C = plasma concentration, C0 = plasma concentration at time zero, t = time after dosing, and k = elimination rate constant.


Total body clearance = VD × k or alternatively, Total body clearance = dose/AUC where the dose is administered intravenously. The plasma level and half-life are also important parameters when the exposure to a toxicant is chronic. Thus, if the exposure is shorter than the half-life, the toxicant accumulates in the body, whereas if the half-life is very short compared to the exposure, the toxicant does not accumulate in the body. It is therefore important to measure the plasma concentration of the toxicant for an assessment of chronic toxicity.

2.9

METABOLISM/BIOTRANSFORMATION OF TOXICANTS

Metabolism is important in a number of body processes, one of which is the detoxification of foreign or exogenous compounds. The biotransformation of a foreign, toxic compound is thus an important aspect of its disposition in vivo. A metabolically unmodified toxicant is often referred to as an active parent compound, and a substance modified by metabolic processes as an active metabolite.

Both types of species may be involved in the manifestation of toxic responses. Almost any reactive chemical that is administered to or ingested by the organism is almost immediately subjected to mechanisms that may confine its translocation within the organism or terminate its existence as a free chemical (Figure 2.9). Upon absorption, a toxicant thus begins changing location, concentration, or chemical density. It may be transported independently by several components of the circulatory system, be absorbed by various tissues or stored, effect an action, be detoxified, or be activated; the parent compound or its metabolite(s) may react with body constituents, be stored, or be eliminated. The study of kinetics, known as pharmacokinetics or toxicokinetics, involved in these processes is a highly specialized branch of toxicology. During the metabolic phase, an active parent compound can be present in blood, liver, or extrahepatic tissues (nonliver tissue), and in the latter two, it may be converted to an inactive metabolite or metabolites. An inactive parent metabolite may produce a toxic metabolite or metabolites in the liver or in extrahepatic tissue; in both these locations, a toxic metabolite may be changed to an inactive form. Therefore, metabolism of a toxicant involves a number of pathways by which the compound is converted to a toxicant or to a substance that is eliminated from the biological system. One of the main results of such metabolic transformation thus is the facilitation of the removal from the body

Figure 2.9 Schematic representation of the pathways through which a toxicant or exogenous chemical may pass during its presence in humans.


of toxic compounds, that, unless excreted, would accumulate to toxic levels. The types of biotransformations are many and varied, and the metabolic systems involved are necessarily very flexible and nonspecific. These are described in detail in Chapter 5. The major factor determining the route(s) of biotransformation is the structure of the compound itself. The elimination of the toxicant from the body is the end point for biotransformation. Kidneys are the main excretory organ of the body for foreign chemicals, and they are most efficient at eliminating polar molecules. Thus, metabolic processes that are detoxifying in nature generally involve reactions that convert nonpolar molecules into more polar ones. In most cases, these changes are advantageous to the body, but in some instances, the process converts basically nontoxic nonpolar compounds into more toxic polar (or more polar) metabolites. The nonpolar lipid-soluble compounds are generally reabsorbed from the kidney tubules or simply equilibrate between plasma and bile by passive diffusion, to no effect. Thus, metabolism may not necessarily be a detoxification process. Its primary purpose is often to facilitate elimination of the compound and alter its biological activity. In some cases, the effect of metabolism might just be to alter elimination from the urinary to the biliary route, for example, by increasing the molecular weight. Metabolism or biotransformation is therefore an important determinant of the activity of a compound, the duration of this activity, and the half-life of the compound in the body. Some very lipid-soluble compounds, such as chlorinated hydrocarbons, polychlorinated and polybrominated biphenyls, and aflatoxins, which are poorly absorbed, may have whole-body half-lives measured in months or even years. The chemical alterations that are the basis of biotransformation of foreign compounds are catalyzed by a number of enzymes, depending on the chemical structure of the compound in question. The most important is the cytochrome P-450 monooxygenase system, but numerous other enzymes may be utilized, both those involved in the intermediary metabolism and those whose main function is the metabolism of xenobiotics. Specific enzymes that recognize particular types of molecules are normally present in small quantities, and the body produces more of them when the need arises, e.g., after a significant exposure to the appropriate foreign molecule. Unfortunately, this process is not easily reversible, and the body does not revert to its preexposure status very rapidly. Thus exposure to one chemical of a particular type may lead to the presence of a large number of specific enzymes when a subsequent exposure to the same or a similar chemical occurs. This process of increasing

the enzyme levels is called enzyme induction. In general, this is beneficial since it helps the body to respond rapidly to repeated exposures. Of course, if the metabolic processes resulting from the response lead to more toxic rather than less toxic metabolites, this induction is counterproductive. Although the major organ involved in the biotransformation of exogenous compounds is the liver, other tissues and organs may be involved to a greater or lesser extent. The importance of liver in this respect relates to its position as a portal to the tissues of the body. By metabolizing and hence removing toxic substances ingested orally and absorbed via the hepatic-portal circulation, the liver protects the organism. In some cases, this metabolic conversion during the absorption phase is almost complete, resulting in a first-pass effect. The gut wall may also carry out biotransformation and, hence, be responsible for a first-pass effect, as may the lung for compounds absorbed by inhalation.

2.10 STORAGE OF TOXICANTS Toxicants are often concentrated in specific tissues in the body. Whereas some achieve the highest concentration at their site of action (e.g., carbon monoxide in hemoglobin), others are concentrated at sites other than the site of toxic action. For example, lead is stored in bone, whereas the symptoms of lead poisoning are due to lead in the soft tissues. The compartment where the toxicant is concentrated but not involved in the toxicological response can be thought of as a storage depot. These can be considered as protective mechanisms, preventing the accumulation of high concentrations of toxicants at the site of toxic action. These toxicants in these depots are always in equilibrium with free toxicant in plasma, and as they are biotransformed or excreted from the body, more is released from the storage site (Klaassen 1986b). The biological half-life of stored toxicants thus can be very long. The following are the major sites of storage for toxicants. 2.10.1

Plasma Proteins

Several proteins in the plasma have the ability to bind to exogenous chemicals as well as some normal physiological constituents (Table 2.4). The majority of foreign chemicals that are bound to plasma proteins are bound to serum albumin. It is the most abundant protein in plasma and serves as a depot protein and transport protein for several endogenous and exogenous compounds. Transferrin, a β1-globulin, is important for transport of iron in the body.


Table 2.4 Examples of Plasma Proteins That Serve as Storage Depots for Physiological Constituents and Toxicants Protein

Physiological constituent/toxicant

Albumin

Transferrin Ceruloplasmin α- and β-Lipoproteins

Immunoglobulins (γ-globulins) α1-Acid glycoprotein

Calcium, copper and zinc ions, bilirubin, uric acid, vitamin C, adenosine, tetracyclines, chloramphenicol, digitonin, fatty acids, suramin, quinocrine, penicillin, salicylate, sulfonamides, streptomycin, acid dyes, phenol red, histamine, triiodothyronine, thyroxine, barbiturates Iron Copper Lipid-soluble vitamins, cholesterol, steroid hormones, vitamin B12, sialic acid, thyroxine Specific for individual antigens Basic compounds

Ceruloplasmin, which carries most of the copper in the serum, is the other metal-binding protein in plasma. The α- and β-lipoproteins are very important in the transport of lipid-soluble compounds, such as vitamins, cholesterol, and steroid hormones. The γ-globulins are antibodies that interact specifically with antigens. Compounds possessing basic characteristics often bind to α1-acid glycoprotein (Klaassen, 1986b; Wilkinson, 1983). Several relevant aspects of binding of toxicants to plasma proteins were described in conjunction with the distribution of toxicants in a preceding section in this chapter. 2.10.2

Liver and Kidney

Liver and kidney have a high capacity to bind chemicals. These two organs also concentrate more toxicants than other organs, primarily because both are important to the elimination of toxicants from the body. 2.10.3

Intracellular Binding Proteins

Within the liver and kidney, several intracellular proteins are important in concentrating the toxicants. Examples include Y protein or ligandin, which has a high affinity for many organic acids; azo dye carcinogens and corticosteroids, in the cytoplasm of the liver; and the cadmiumbinding protein metallothionein, found in the kidney and liver (Levi et al., 1969; Litwalk et al., 1971; Klaassen and Shoeman, 1974).

2.10.4

Fat

Since most toxicants are lipophilic in nature, they readily distribute and concentrate in body fat. Toxicants appear to accumulate in fat by simple physical dissolution in the neutral fats. Thus, a toxicant with a high lipid/water partition coefficient may be stored in the body fat to a large extent. Although such storage lowers the concentration of the toxicant in the target organ and thus may serve as a protective mechanism, intoxication can result from short-term starvation (Klaassen, 1986b). Examples of well-known toxicants stored in this way include pesticides such as chlordane and DDT and the polyhalogenated aromatics, such as polychlorinated and polybrominated biphenyls. 2.10.5

Bone

Bone can also serve as a reservoir for such compounds as fluoride, lead, and strontium. It is also a major storage site for some toxicants. Toxicants deposited in bone are not irreversibly sequestered and thus can be released by ionic exchange at the crystal surface and by dissolution of bone crystals through osteoclastic activity (Klaassen, 1986b). Compounds that accumulate in the body as a result of repeated frequent ingestion as contaminants of food have occasionally resulted in insidious harm to sizable populations of humans. Methylmercury and lead poisoning instances due to either misuse as food or industrial discharge of the compound are well documented in the literature.

2.11 EXCRETION OF TOXICANTS One important factor in the toxicity of foreign compounds is excretability. The more rapidly they are eliminated, the less likely they are to exert an adverse effect. If, on the other hand, their retention in the body is prolonged, the potential for toxic effects is greater. Although there are a wide variety of synthetic organic chemicals of recent origin, relative to the evolutionary time scale, they all can be eliminated from the body without special physiological systems. Most exogenous chemicals, however, are not readily eliminated until they are in a form similar to that utilized for the elimination of endogenous substances (Guthrie and Hodgson, 1987b). In general, a similar trend of metabolic reactions that yield metabolites of lower toxicity as a result of improved excretability (Figure 2.10) is seen. This trend is not without exceptions. For instance, the completely nonpolar molecules, such as methane and ethane, often are inert biologi-


Figure 2.10 A generalized trend of biotransformation reactions that yield metabolites of lesser toxicity as a result of improved excretability.

cally and require no detoxification. However, if a nonpolar molecule is biologically active (e.g., benzene, carbon tetrachloride), it is not likely to be readily detoxified and is therefore very toxic. There are a number of routes of excretion, the major route is the kidneys for most nongaseous or nonvolatile compounds. In fact, more chemicals are eliminated from the body by this route than any other. The liver and the biliary system are also important. In contrast, routes of elimination such as milk, lungs, alimentary excretion, sweat, tears, hair, and semen are generally of minor importance. The major routes of excretion of foreign compounds are now briefly described. 2.11.1

Renal/Urinary Excretion

The kidneys are primarily excretory organs. In addition to removing most by-products of normal metabolism, kidneys are also the primary organ for excretion of polar xenobiotics and hydrophilic metabolites of any lipophilic toxicants that the body has encountered. The toxicants are transported to the kidneys either by solubilization in blood or by binding to plasma proteins. Toxic substances and other foreign compounds are removed from the blood as it passes through the kidneys; the blood flow represents about 25% of the cardiac output. The physicochemical principles governing the excretory processes are essentially as previously described for ab-

sorption. Three processes are primarily responsible for the elimination of the toxic substances from the body: passive glomerular filtration, passive tubular diffusion, and active tubular secretion. Glomerular Filtration The blood (plasma) is first passively filtered at the glomerulus and the filtrate passed into the tubules, where reabsorption may take place for lipid-soluble, nonionized compounds. Filtration at the glomerulus normally occurs for most compounds of molecular weight less than 70,000, as the pores in the membrane are relatively large (70–100 Å). Only the non-protein–bound forms of compounds are filtered. The concentration of the compound in the glomerular filtrate therefore approximates that in the plasma in the unbound form. Compounds too large to pass, those bound to proteins, and nonionized and very lipid-soluble compounds, are reabsorbed into the bloodstream by passive diffusion. These are subsequently eliminated by other avenues. Passive Tubular Diffusion Toxicants can also be excreted from the plasma into urine by passive diffusion through the tubule. The pH of the urine is an important determinant factor in this regard. Therefore, bases are more readily excreted if the urine is acid, and vice versa for the excretion of acids. Generally, weak acids are frequently biotransformed to stronger acids, thereby increasing the percentage in the ionic form. Active Tubular Secretion Active tubular secretion is an important mechanism of elimination for ionized compounds. It is not affected by plasma protein binding of the compound. This is because the mechanism is very rapid and not concentration dependent. Therefore, the dissociation of the protein-bound compound continuously provides more compound for active transport. Organic acids and bases appear to be transported by different secretory processes, which are located in the proximal convoluted tubules. Because it is energy dependent, this process can be inhibited by metabolic inhibitors or competitively by other organic acids or bases. Factors affecting kidney function, such as age and disease, may have a marked effect on the toxicity of compounds excreted into the urine. 2.11.2

generally involves active secretion rather than passive diffusion, and there appear to be specific transport systems for organic acids, organic bases, and neutral compounds. Quaternary ammonium compounds may be actively secreted into the bile by a separate process. This route of excretion seems to apply particularly to comparatively large, ionized polar molecules (Timbrell, 1982; Klaassen, 1986b). The liver is indeed in a very advantageous position for removing toxicants from blood after they are absorbed from the GI tract. The blood from the GI tract must first pass through the liver before reaching the general systemic circulation. Thus, liver can remove compounds from blood and prevent their distribution to other parts of the body. The liver is also the main site of biotransformation of toxicants. Thus, compounds can be metabolized and conjugated rapidly in the first pass through the liver and then directly excreted into the bile. Such biliary excretion obviates the need for metabolites to enter the bloodstream for excretion by the kidneys. A toxicant may be excreted by liver cells into bile and thus pass into small intestine and remain there. The excretion of compounds via the bile into the intestine may lead to the reabsorption of the compound if intestinal conditions permit. The most common mechanism for reabsorption is hydrolysis or metabolism of conjugates of the compound by intestinal microflora. The hydrolyzed conjugate, which is less polar, can be absorbed by the intestine and returned to the liver through the portal or enterohepatic circulation (Figure 2.11). This process may have toxicological consequences, especially if the metabolite is more toxic than the excreted conjugate, or if it prolongs the half-life of the compound. Enterohepatic

Hepatic/Biliary Excretion

Hepatic/biliary excretion is the second most significant route of elimination of toxicants from the body. This route


Figure 2.11 Enterohepatic circulation. Circulation of the compound is indicated by arrows. (From Timbrell [1982].)

circulation can be recognized by examination of the profile of the plasma concentration of the toxicant. The plasma concentration does not show a smooth decline but rather increases after various intervals when reabsorption has taken place. High-molecular-weight conjugates are examples of compounds that undergo biliary excretion. The molecular weight of the compound, its charge, and the animal species influence biliary excretion of toxicants. A molecular weight >300 is generally a prerequisite for biliary excretion. Some compounds are almost exclusively eliminated from the body by biliary excretion, and consequently toxicity may be markedly increased if bile secretion is impaired. Inhibition of the detoxifying monooxygenase enzyme system, sex, and age also influence biliary excretion. The hepatic excretory system is also not fully developed in the newborn. This is why some compounds are more toxic in newborns than in adults (Klaassen, 1972, 1973). Alternatively, the development of the hepatic excretory mechanism can be promoted by administering microsomal enzyme inducers (Klaassen, 1974). 2.11.3

Lungs/Pulmonary Excretion

Gases and volatile compounds are usually eliminated from the body by the pulmonary route, as are the volatile metabolites of nonvolatile compounds. The rate of elimination depends on solubility in blood, rate of respiration, and blood flow to the lungs (Guthrie and Hodgson, 1987b). Pulmonary excretion takes place by simple diffusion, but generally it is very rapid. The best-known compounds subject to respiratory elimination are ethanol, anesthetic gases, pesticide fumigants, and many volatile organic solvents, such as ether. 2.11.4

Gastrointestinal Tract

Many toxic compounds are excreted in feces. Appearance in feces is generally due to the following factors (Klaassen, 1986b): 1. 2. 3. 4.

The chemical was not completely absorbed after oral ingestion. It was excreted into the bile. It was secreted in saliva, in gastric or intestinal secretory fluid, or in pancreatic secretion. It was secreted by the respiratory tract and then swallowed.

Toxicants can enter the lumen of the GI tract via passive diffusion. Intestinal excretion may be a major route of elimination of highly lipophilic compounds, such as organochlorine pesticides, 2,3,7,8-tetrachlorodibenzodioxin


(TCDD), and the polychlorinated biphenyls (Rozman et al., 1982). The GI elimination of these compounds, each of which has a very long biological half-life, can be enhanced by increasing the lipid composition of the diet. 2.11.5

Excretion via Milk, Sweat, and Saliva

These are minor routes of excretion for some compounds. Of these, the secretion of toxic compounds into the milk is extremely important because (a) a toxic material may be passed in milk from mothers to the nursing child, and (b) compounds can be passed from cows to humans by this route (Klaassen, 1986b). Because milk is more acidic (pH ~ 6.5) than plasma, basic compounds may be concentrated in milk. Furthermore, because milk has high fat content (3%–5%), lipophilic compounds, such as DDT and polychlorinated and polybrominated biphenyls, are concentrated in it, thus forming a major route of their excretion. Toxic compounds excreted into sweat may produce dermatitis; those excreted in saliva are usually swallowed and are then available for GI absorption. Excretion via these three minor routes is dependent on diffusion of the nonionized lipid-soluble form of the toxicant.

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3 Manifestations of Organ Toxicity

3.1

INTRODUCTION

The biochemical events following exposure to a toxic agent generally result in clinical manifestations or symptoms. These symptoms may be characteristic of the particular physiological process or organ system thus affected. Often multiple symptoms associated with the toxicological events indicate that more than one system is affected. Virtually all physiological processes and organ systems in the body are subject to toxic effects. Thus one must understand that all these symptoms are interrelated, so that toxic injury to the primary target may have repercussions in other systems. This is particularly true of damage to the central nervous and hematopoietic systems (Concon, 1988). Although, for the sake of convenience, the toxic effects are divided into acute, subacute, and chronic on the basis of time course, it is often not possible to categorize each type of toxicity or the effects of individual chemicals according to one of these three classifications. For example, lung toxicity may be an acute, subacute, or chronic effect. Similarly, a particular chemical may produce an acute effect at one exposure level and a subacute or chronic effect at another. In spite of these classification difficulties, it has been common practice to identify the different types of toxicity with one of the three time course categories. Thus organ damage has usually been classified as an acute or subacute effect, and most other types of toxicity have been assigned to the chronic classification. The former category includes neurotoxicity, hepatotoxicity, nephrotoxicity, hematotoxic-


ity, skeletal toxicity, reproductive toxicity, and immunotoxicity. The toxic effects that are most relevant to food toxicants and are generally included in the chronic category are allergenicity, mutagenicity, teratogenicity, and carcinogenicity. This is because these effects, in many cases, may be induced by toxicants at the very low levels that are found in food. However, organ toxicity effects may be induced by small quantities of toxicants. Organ toxicity has generally been thought of as an acute or subacute effect for a number of reasons (Kamrin, 1988). One is that the effect often occurs soon after the initiation of exposure, especially at high doses. In addition, the degree of toxicity reflects the frequency and intensity of exposure in the traditional dose-response fashion. Another characteristic of organ toxicity is that the organ that is affected is directly linked to the type of chemical to which exposure has occurred. In this chapter, several important manifestations of organ toxicity are briefly described. The chronic manifestations of carcinogenesis, mutagenesis, and teratogenesis are described in Chapter 4.

3.2

NEUROTOXICITY

The brain and the central nervous system (CNS) are protected from toxicants by the blood-brain barrier. This barrier, which normally protects the brain from toxicants that can damage other soft tissues, however, does not exclude all toxic substances. Furthermore, several areas of the nervous system lack the barrier. These include the median

eminence with arcuate nucleus (Reese and Brightman, 1968), median preoptic region (Brightman and Reese, 1969), choroid plexus, and area postrema (Olsson and Hossman, 1970), all these belonging to the CNS, and the dorsal root ganglia (Brierly, 1955) and autonomic ganglia (Jacobs, 1977) of the peripheral nervous system (PNS). These areas are therefore more susceptible to some toxic compounds than other areas of the CNS and PNS, which have greater resistance. In addition to such differences in the morphological characteristics of the nervous system, even those toxic substances that can penetrate brain tissue do not affect equally all of the cell types in the brain. Different brain areas usually have different sensitivities to toxicants, reflecting unique biochemical properties of the cells as well as differences in degree of vascularization of brain areas (Norton, 1986). Toxic responses of the CNS may be manifested in two ways: structural toxicity and functional toxicity. 3.2.1 Structural Toxicity When CNS cells are damaged by exposure to toxic chemicals either by direct contact or by secondary effects such as anoxia subsequent to diminished oxygen supply, some similar effects are observed. These effects are the swelling of the cell and cytoplasmic organelles, dispersion of the rough endoplasmic reticulum, and swelling of the nucleolus (Norton, 1986, Stine and Brown, 1996). The changes are accompanied by decreases in the cytoplasmic pH, in activity of the oxidative enzyme systems, and in the synthesis of protein and other cell components. Certain cells are more sensitive to anoxia than others. The sequence of vulnerability to neurotoxicants can be described as neurons > oligodendrocytes > astrocytes > microglia > cells of the capillary endothelium. The lack of oxygen to brain and the CNS may result from one of the three types of anoxia: anoxic, ischemic, and cytotoxic. These conditions result from a failure in blood oxygenation, a decreased blood supply, or interference in oxygen transport, cellular metabolism, or respiration. Anoxic Anoxia Anoxic anoxia results from a primary lack of oxygen even in the presence of adequate blood flow. Such a primary condition can result from either direct interference with respiration by toxic substances or interference with the oxygen-carrying capacity of the blood. If respiration is restored before cardiovascular failure occurs, neurons in the CNS that are sensitive to anoxia may be destroyed without death of the organism. Examples include the production of


carboxyhemoglobin by carbon monoxide and of methemoglobin by nitrites. Failure in blood oxygenation may also follow from respiratory paralysis by paralytic agents such as botulinum exotoxin and saxitoxin. Ischemic Anoxia Ischemic anoxia results from a decrease in arterial blood pressure to a level below that which supports the brain adequately with oxygen (Norton, 1986). The stagnation of the blood in the brain leads to an inadequate supply of needed substances and an accumulation of metabolic byproducts such as lactic acid, ammonia, and inorganic phosphate. Cardiac arrest caused by toxic substances is one obvious cause of inadequate blood flow. Severe hypotension that results from vasodilatation can also diminish the brain’s blood supply considerably, as in the effect of cyanide poisoning on the heart. The rupture of critical blood vessels in the brain, which results in hemorrhage or thrombosis, may also cause local ischemic anoxia of the brain. Cytotoxic Anoxia Cytotoxic anoxia is a consequence of interference with cell metabolism in the presence of an adequate supply of both blood and oxygen. In contrast to the great susceptibility of the neurons of the adult brain to ischemic and anoxic anoxia, it is the oligodendroglia that are more susceptible to this type of injury caused by metabolic inhibitors such as cyanide, methionine sulfoximine, azide, dinitrophenol, and malononitrile. Cytotoxic anoxia may also result from hypoglycemia, produced by an excess of insulin. Hypoglycin A from the akee fruit is an example of such a metabolic inhibitor. 3.2.2 Functional Toxicity An exposure to neurotoxicants may also result in functional toxicity of the CNS. Five types of central and peripheral nervous system function and the associated toxic manifestations are shown in Figure 3.1. For example, sensory loss is exemplified by blindness; deafness; and loss of the sensation to touch, temperature, pressure, and pain; and paresthesia. The loss of motor functions is associated with general paralysis (paresis) and muscle incoordination (ataxia). Functional neurotoxicity is also associated with signs suggesting loss of integrative functions, such as loss of memory and learning ability, seizures, convulsions, excitation, hyperkinetic behavior, depression, and coma. Many types of behavioral and emotional alterations and mental retardation are also manifestations that are definitely associated with neurotoxic effects (Wiener, 1970; Norton, 1986; Levi, 1987; Kamrin, 1988; Stine and

Figure 3.1

Central and peripheral nervous system functions and associated toxic manifestations.

Brown, 1996; Niesink et al., 1996). Examples of these effects are those associated with chronic lead poisoning. 3.2.3 Neurotoxicants Several classifications have been suggested for neurotoxicants (Scholz, 1953; Malamud, 1963; Windle, 1963; Brucher, 1967; Brierley et al., 1971). However, no one classification is ideal since a chemical may have more than one effect and the susceptibilities of the nervous system of different animal species can hardly be considered identical. Furthermore, any classification is subject to some error since it depends on data that are at best fragmentary. The following classification is based on the suggestions of Norton (1986), who classified the neurotoxicants according to their primary toxic action: 1.

2.

Agents causing anoxia: These chemicals cause anoxic damage to gray matter (neurons and astrocytes) with variation in pattern of damage depending on which of the three types of anoxia is produced. Examples include barbiturates, carbon monoxide, cyanide, azide, and nitrogen trichloride poisoning. Agents damaging myelin: These chemicals cause damage to myelin, affecting oligodendro-


3.

4.

5.

cytes or Schwann cells, resulting in encephalopathy if central white matter is involved or polyneuritis if peripheral cells are damaged, e.g., isonicotinic acid hydrazide (INH, isoniazid), triethyltin, hexachlorophene, lead, thallium, and tellurium. Agents causing peripheral axonopathies: These are substances with a predilection for causing damage to axons of peripheral neurons. Examples of this type of neurotoxicants include alcohol, acrylamide, carbon disulfide, hexanedione, bromophenylacetylurea, and organophosphorus compounds. Agents causing primary damage to perikarya of peripheral neurons: Some neurotoxicants, such as organomercurials, vinca alkaloids (vincristine and vinblastine), and iminodipropionitrile, cause primary damage to the perikaryon, the main site of synthesis of protein of peripheral neurons. Neurotoxicants causing damage to synaptic junctions of the neuromuscular system: The synaptic clefts and the terminals of myelinated axons are uniquely vulnerable to toxic chemicals that cause severe poisoning in humans. Several well-known toxins, including botulinum toxin, tetrodotoxin, saxitoxin, batrachotoxin, organo-

6.

3.3

chlorine pesticides, pyrethrins, and lead, belong to this category of neurotoxins. Agents causing localized CNS lesions: The compounds in this group cause lesions restricted in distribution, which primarily affect localized anatomical areas in the CNS. These include mercury, lead, manganese, excessive quantities of glutamate and aspartate, methionine sulfoximine, gold, thioglucose, organotin and organochlorine pesticides, and certain nutrient antagonists. Such antinutritives are antimetabolites, antiniacinamides, and antihistamine factors.

LUNG TOXICITY

The principal function of the lungs is gas exchange, providing oxygen to the tissues and removing carbon dioxide. Lung as an organ is in a particularly vulnerable position as regards toxic substances. It may be involved in both absorption and excretion of volatile toxins. It may also suffer damage from nonvolatile toxic compounds administered by other routes. Although many different toxicants may damage the lung, the patterns of cellular injury and repair are relatively constant. One of the most obvious and familiar is irritation caused by volatile compounds such as ammonia and chlorine. A severe or persistent irritation may lead to constriction of airways and edema of lung tissue. Another wellknown symptom of lung toxicity is fibrosis, a formation of collagenous tissue caused by substances such as silica (silicosis) and asbestos (asbestosis). In addition, numerous agents, including microorganisms, spores, dust, and chemicals, are known to elicit allergic responses. Perhaps the most severe response of the lung to injury is cancer, the primary causative agent of which is inhaled cigarette smoke. Lung toxicity, however, is not of much relevance to the field of food toxicology, except perhaps in relation to workers involved in pesticide spraying of food crops.

3.4

cytochrome P-450-dependent monooxygenase system. Although most biotransformations are detoxification reactions, many oxidative reactions produce reactive metabolites that can produce lesions within the liver. Such damage is often seen in the centrilobular region, which also has higher concentrations of cytochrome P-450. 3.4.1 Types of Liver Injury On the basis of guidelines promulgated by the U.S. Public Health Service for the detection of hepatotoxicity due to drugs and chemicals, the hepatic lesions can be divided into two categories as follows (Davidson et al., 1979): Type I lesions: These are predictable, are dose- and time-dependent, and occur in most, if not all, subjects exposed to appropriate doses of the causative agent. These lesions are usually readily reproducible in animals. Type II lesions: These are nonpredictable, are doseand time-independent, occur sporadically, and often become apparent only after monitoring of a large number of exposed individuals. These lesions are usually not reproducible in animals. This classification, however, does not take into consideration the actual morphological characteristics of the liver injury. For a morphological classification, the system proposed by Popper and Schaffner (1959) is still widely used. It describes five groups of reactions: 1.

2.

HEPATOTOXICITY

The liver, the largest organ in the body, is often the target organ for chemically induced injuries. Several important factors are known to contribute to the liver’s susceptibility. First, compounds absorbed in the GI tract are transported by the hepatic portal vein to the liver (see Chapter 2). Thus the liver is the first organ perfused by chemicals absorbed in the gut. A second factor is a high concentration in the liver of xenobiotic-metabolizing enzymes, primarily the


3.

Zonal hepatocellular alterations without inflammatory reaction: Chemicals in this category all produce zonal changes, either necrosis or fat accumulation. This type of lesion is probably the best understood type of hepatic injury (a type I lesion). It is dose dependable and predictable and can be easily reproduced in several animal species. Intrahepatic cholestasis: This category contains drugs of unrelated chemical structures that produce a type of jaundice resembling that produced by extrahepatic biliary obstruction in a very small percentage of the population. Inflammation or blocking of the bile ducts results in retention of bile salts as well as accumulation of bilirubin, which causes jaundice. There is no relationship between dose and response, and production of lesions in animals is not possible (a type II lesion). Hepatic necrosis with inflammatory reaction: This type of liver injury is characterized by pro-

4.

5.

gression to a massive necrosis characteristic of viral hepatitis. This is also a type II lesion. Unclassified group: This category contains a variety of hepatic injuries that do not fit into any type of scheme. For some, the lesions are associated with manifestations of abnormality in several other organs. Hepatic cancer: A number of chemicals are now being recognized as hepatocarcinogens in animals. The most common type of primary liver tumor is hepatocellular carcinoma; other types include cholangiocarcinoma, angiosarcoma, glandular carcinoma, and undifferentiated liver cell carcinoma. However, only a few hepatocarcinogens, such as vinyl chloride, which causes angiosarcoma, are human carcinogens.

Unless they occur on a massive scale, necrotic lesions due to cell death are not necessarily critical because of the regenerating capability of the liver. Cell necrosis is preceded by a number of morphological changes such as cytoplasmic edema, dilatation of endoplasmic reticulum, disaggregation of polysomes, accumulation of triglycerides, swelling of mitochondria with disruption of cristae, and dissolution of organelles and nucleus (Levi, 1987; Plaa, 1986; Niesink et al., 1996). Biochemical events that may lead to these changes include binding of reactive metabolites to proteins and unsaturated lipids (including lipid peroxidation and subsequent membrane destruction), disturbance of cellular Ca2+ homeostasis, interference with metabolic pathways, shifts in Na+ and K+ balance, and inhibition of protein synthesis. Fatty liver results from an abnormal accumulation of fats, mainly triglycerides, in the parenchymal cells. Excess lipid can result from oversupply of free fatty acids from adipose tissues or, more commonly, from impaired release of triglycerides from the liver into plasma (Plaa, 1986; Levi, 1987; Stine and Brown, 1996). The importance of fatty liver in liver injury is not clearly understood, and fatty liver in itself does not necessarily indicate liver dysfunction. Another type of liver injury that has received great attention is cirrhosis. This progressive disease is characterized pathologically by the presence of collagen throughout most of the liver. Cirrhosis is often associated with liver dysfunction and frequently results in jaundice. In humans, chronic use of alcohol is the single most important cause of cirrhosis. Well-known examples of hepatotoxic agents and associated liver injury are presented in Table 3.1.


Table 3.1 Examples of Hepatotoxic Agents and Associated Liver Injury Liver necrosis Acetaminophen, allyl alcohol, beryllium, bromobenzene, furosemide, thioacetamide Fatty liver Allyl formate, cerium, cycloheximide, emetine, ethanol, thionine, mitomycin C, puromycin, tetracycline Both liver necrosis and fatty liver Aflatoxins, azaserine, carbon tetrachloride, chloroform, dimethylnitrosamine, galactosamine, phosphorus, pyrrolizidine alkaloids, tannic acid, tetrachloroethane, trichloroethylene Cholestasis (drug-induced) Promazine, amitriptyline, diazepam, estradiol, sulfanilamide, mepazine acetate, phenindione, methimazole Hepatitis (drug-induced) Iproniazid, isoniazid, imipramine, colchicine, halothane, methyldopa Carcinogenesis (experimental animals) Aflatoxins, pyrrolizidine alkaloids, safrole, cycasin, polychlorinated biphenyls, vinyl chloride, urethane Source: Compiled from Levi (1987), Plaa (1986), and Zimmerman (1978).

3.5

NEPHROTOXICITY

The kidneys have a greater likelihood of toxic injury from ingested substances than any other organ in the body. Several factors may be involved in this sensitivity, but perhaps the most important is high renal blood flow (Levi, 1987; Timbrell, 1982; Hook and Hewitt, 1986; Stine and Brown, 1996). Although the two kidneys constitute 100 kGy) of irradiation, and breakdown of lipids into hydrocarbons, aldehydes, esters, and ketones (Table 9.11). Generally, the removal of oxygen during irradiation inhibits the oxidation of lipids. This can be achieved by packaging the food or oil in an oxygen-free container or by irradiating under vacuum. The chemical changes that occur in lipids as a result of irradiation can also be minimized by applying the treatment to frozen samples. Vitamins In general, vitamins exposed to levels of irradiation show some losses due to destruction. Additionally, antioxidant vitamins, such as vitamin C and E, can combine with free radicals and lose their vitamin activity. Alternatively, free radicals and their products can attack and destroy vitamin structure or activity. However, certain vitamins have been found to be quite resistant to irradiation-induced destruction; these include vitamin B12, folacin, and pantothenic acid. In contrast, the water-soluble vitamins thiamine and ascorbic acid are the least resistant to effects caused by irradiation. From the available literature data it is clear that


Formation of flavored compounds

Cross-linking Denaturation Activation, deactivation, or no effect Altered water-binding capacity Altered gelling, emulsifying, and foaming properties Formation of flavored compounds Activation, deactivation, or no effect Production of rancidity due to oxidation in the presence of oxygen Production of rancidity due to oxidation in the presence of oxygen Production of rancidity due to oxidation in the presence of oxygen Destruction of polyunsaturated fatty acid

vitamins are sensitive to degradative loss due to irradiation, just as they are to heating and other processes. The magnitude of sensitivity varies from vitamin to vitamin, product to product, and treatment condition to treatment condition. In order to retain vitamins in foods that are to be irradiated, low treatment doses combined with cold sample temperatures plus oxygen and light exclusion are often recommended. 9.9.3

Effect on Microorganisms

Most of the applications of food irradiation aim at certain biological effects, such as destruction of microorganisms (i.e., those causing food spoilage and/or those causing disease in humans). It is now universally accepted that the DNA in the chromosomes is the most critical target of ionizing radiation. Effects on the cytoplasmic membrane may play an additional role in some circumstances (Grecz et al., 1983; Diehl, 1995). Destruction of microorganisms by irradiation is affected by several factors. Microorganisms differ in their sensitivity to irradiation, depending on morphological variation, just as they differ in their sensitivity to heating, drying, and freezing. Generally, the order of resistance varies, with viruses > bacterial spores > bacterial cells >

yeasts and molds. Generally, the more simple the lifeform, the more resistant it is to irradiation. The resistance to irradiation is expressed as the D10 value, or dose required to reduce the microbial population in a given medium (or food) 10-fold. Thus, an organism with a D10 value of 0.5 kGy is more resistant to irradiation than one with a D10 value of 0.25 kGy, since twice the dose is required to destroy the same number of cells in the population. The D10 values for selected nonsporogenic and spore-forming bacteria are summarized in Table 9.12. No public health hazard related to microorganisms will arise from high-dose irradiation because this process results in a commercially sterile product. In contrast, when foods are treated with nonsterilizing doses of radiation, some microorganisms do survive. This finding has raised a number of concerns (HMSO, 1986, WHO, 1994; Diehl, 1995; Nawrot et al., 1999): 1.

2.

3.

4.

5.

Enhanced selective effects of radiation on the microbial flora in foods: harmless organisms are less resistant to radioactivity than certain pathogenic species. Therefore, more pathogenic microorganisms may survive. It is also possible that spoilage organisms may be preferentially destroyed by irradiation, thereby allowing pathogenic organisms such as Clostridium botulinum, C. perfringens, and Bacillus cereus to survive and grow unchecked. In the absence of spoilage organisms, the food would then appear fit for consumption on the basis of typical organoleptic properties yet contain increased numbers of pathogens and pose a hazard to human health. Enhanced mutations in the surviving population: this may convert nonpathogenic organisms into more virulent strains. Increased radiation resistance by repeated sublethal treatment with radiation: radiation resistance of certain microorganisms has been observed under specific laboratory conditions. Potential change in diagnostic characteristics of microorganisms as a result of irradiation: the species or strain may not be correctly identified. Enhanced toxin formation in toxin-producing bacteria or molds: increased production of aflatoxin has been reported when spores of Aspergillus flavus or A. parasiticus or cultures derived from such spores were irradiated.

Several studies reviewed by Diehl (1995) and Nawrot and associates (1999) show that microbiological safety of irradiated food is comparable with that of food preserved by other acceptable preservation methods, and


that there is no indication of specific bacteriological hazards associated with radiation processing. On the basis of the existing body of scientific evidence, a WHO panel of experts concluded that there is no reason to suppose that irradiated food needs to be subjected to controls different from those regularly applied to food processed by conventional techniques (WHO, 1994). 9.9.4 Effects on Packaging Materials With the exception of such applications as sprout inhibition in potatoes or onions, insect disinfestations in bulk grain, or delay of postharvest ripening of fruits, irradiation of foods is usually carried out with packaged food items. The reasons for this include prevention of microbial reinfection or insect exposure, prevention of water loss, exclusion of oxygen, prevention of mechanical damage during transport, and improved handling and marketing (Diehl, 1995) The packaging material used must not release radiation-induced reaction products or additives to the food; nor should it lose its functional qualities such as mechanical strength, seal stability, or impermeability to water. Radiation resistance of various packaging materials has been extensively tested. Some are more radiation-resistant than others, but in the dose range of up to 10 kGy, almost all materials commonly used for food packaging are suitable because radiation effects are minor in this range. In the United States, packaging materials used in the irradiation of prepackaged foods must be approved for this purpose by the FDA. 9.9.5 Applications The application of radiation may be divided broadly into three categories: high-dose (>10kGy), medium-dose (1–10 kGy), and low-dose (20%), marked cyanosis, fatigue, and dyspnea are observed. At levels over 40%, severe cyanosis, tachypnea, serious cardiopulmonary signs, tachycardia, and depression can occur. Ataxia, coma, and death occur above 60% (Concon, 1988). Most cases of methemoglobinemia involving infants occur because of high levels of nitrates in drinking water or consumption of high-nitrate spinach. The levels of nitrites in food as a derived toxicant are closely related to the levels of nitrates. The average nitrate content of some common foods in the United States and per capita daily intake are summarized in Table 9.14. Many vegetables contain high levels of nitrates. Beets, celery, lettuce, radishes, and spinach may contain more than 600 ppm nitrate nitrogen. These vegetables alone may contribute more than 86% of the total daily intake of nitrates. A large proportion of the total daily intake may also be derived from potatoes because of greater per capita consumption than of any other vegetable, even though potato contains only moderate levels of nitrates. Drinking water, especially that from shallow wells, may also contribute significantly to the level of nitrates in food. Shallow well water may contain as much as 100 mg nitrate/L (Simon et al., 1964). 9.10.2

N-Nitrosamines

As mentioned earlier, nitrates and nitrites can be converted to N-nitroso compounds in vivo. N-Nitroso compounds can be divided into two classes: the nitrosamines and the nitrosamides (and related compounds). Nitrosamines are N-nitroso derivatives of secondary amines, whereas nitrosamides are N-nitroso derivatives of substituted ureas, amides, carbamates, guanidines, and similar compounds (Mirvish, 1975). The general chemical structures of the three general types of nitrosamines significant in food toxicology are shown in Figure 9.11. The most common dialkylnitrosamines are dimethyl- and diethylnitrosamine (DMN and DEN, respectively). Both compounds have been widely studied and are found in foodstuffs. The alkyl group in these compounds may be symmetrical or asymmetrical and may contain

Table 9.14 Average Nitrate Contents of Common Foods in the United States and Per Capita Daily Intake Nitrate, mg/100 g Food

Content

Ingestion

Total vegetables Asparagus Beet Beans, dry Beans, lima Beans, snap Broccoli Cabbage Carrot Celery Corn Cucumber Eggplant Lettuce Melon Onion Peas Peppers, sweet Pickles Potato Potato, sweet Pumpkin/squash Spinach Sauerkraut Tomato and tomato products Breads All fruits Juices Cured meats Milk and milk products Water

1.3–27.6 2.1 276.0 1.3 5.4 25.3 78.3 63.5 11.9 234.0 4.5 2.4 30.2 85.0 43.4 13.4 2.8 12.5 5.9 11.9 5.3 41.3 186.0 19.1 6.2 2.2 1.0 0.2 20.8 0.05 0.071

8609.1 2.8 546.0 10.0 6.6 258.0 127.0 548.0 104.0 1600.0 77.0 7.8 14.8 1890.0 935.0 159.0 19.8 33.5 56.0 1420.0 26.4 38.0 420.0 33.2 198.0 198.0 130.0 10.7 1554.0 25.0 71.0

other functional groups. The parent compounds of dialkylnitrosamines are secondary amines. Nitrosamine formation can occur outside or inside the body; the principal precursors are the various amines and amides and nitrites (Mirvish, 1975, 1983). Thus, the fundamental requirements are a secondary amino nitrogen and nitrous acid. In reality, the nitrosating species is nitrous anhydride or, in the presence of thiocyanate or halides, nitrosylthiocyanate or nitrosylhalide. The precursors to nitrosamine formation occur widely in both the environment and biological systems (Table 9.15 and 9.16). Hence, low-level amounts of nitrosamine have been widely found in many foods and other environments. Nitrosamines can also be formed in vivo. In fact, the conditions in the alimentary tract from the mouth to the


R1 R2

N N

O

Dialkylnitrosamine

R1

N N O C NH NH R2

Acylalkylnitrosamine

R1

N N O C O R2

Nitrosoguanidine Figure 9.11 General structures of three types of nitrosamines found in foodstuffs.

rectum are quite conducive to nitrosamine formation. First, oral bacteria may promote the reduction of nitrate to nitrite, so that the total nitrite load is increased (Tannenbaum et al., 1974, 1976). Second, nitrosamine formation can also be promoted in the mouth by oral bacteria. Third, saliva can promote the formation of cyanamides from secondary amines as well as primary amines. Both normal and hypoacidic conditions in the stomach also favor nitrosamine formation. The normal acidity of the stomach is ideal for nitrosation, whereas hypoacidity may allow some microorganisms to promote nitrate reduction. The dual occurrence of nitrate reduction and nitrosamine formation in human subjects with gastric hypoacidity who are gavaged with sodium nitrate and diphenylamine was reported by Sander and Schweinsberg (1972). The microflora in the small intestine promotes nitrification of ammonia and organic nitrogen compounds. Five strains of Escherichia coli from the human gut have been demonstrated to form nitrosamines from dimethylamine, diethylamine, piperidine, pyrrolidine, and other amines in the presence of sodium nitrite at neutral pH. In addition, strains of Bacteroides, Bifidobacterium, Clostridium, and Enterococcus spp., which do not reduce nitrate, have been shown to form nitrosamines on replacement of nitrate in the reaction medium with nitrite (Hawksworth and Hill, 1971). The nitrosation reaction is dependent on physicochemical factors. One of the most effective inhibitors of

Table 9.15 Nitrosamine Precursors Endogenous or Formed (Derived) in Foodstuffs Compound Creatine, creatinine Trimethylamine oxide Trimethylamine Dimethylamine Diethylamine Sarcosine Choline, lecithin Proline, hydroxyproline Pyrrolidine Piperidine Methylguanidine Citrulline Carnitine Dipropylamine Dibutylamine

Nitrosamine formeda

Food Meats, meat products, milk, vegetables Fish Fish Fish, meats, meat products, cheese Cheese Meats, meat products, fish Eggs, meats, meat products, soybean, corn Meats, meat products, other foodstuffs Meats, meat products, paprika Meats, meat products, cheese, black pepper Beef, fish Meats, meat products, vegetables Meats, meat products Cheese Cheese

NSA DMN DMN DMN DEN NSA DMN Npro, Npyr Npyr Npip MNC NCit DMN DPN DBN

a

NSA, nitrososarcosine; DMN, dimethylnitrosamine; DEN, diethylnitrosoamine; Npro, nitrosoproline; Npyr, nitrosopyrrolidine; Npip, nitrosopiperidine; MNC, methylnitrosocyanamide; DPN, di-N-propylnitrosoamine; DBN, di-N-butylnitrosoamine; Ncit, nitrosocitrulline. Source: From Concon (1988).

tions of cooking and/or processing. It must be noted, however, that the methods used in nitrosamine analysis in food may underestimate the level of these carcinogens. The reason is that, apart from the low recoveries inherent in the methods, determinations using separation of individual nitrosamines automatically exclude many others that may be present. Collectively, the latter may be quite significant. Thus, the amount of total nitrosamines in a food may be more pertinent in assessing the hazard from these carcinogens. Therefore, the methods of analysis should also focus on the determination of total nitrosamines rather than just each individual compound.

nitrosamine formation is ascorbic acid, which reacts with nitrite readily to form nitric oxide and dehydroascorbic acid (Mirvish et al., 1972). It thus competes for any nitrite present and hence reduces the availability of this reactant for nitrosamine formation. Other inhibitors of the nitrosation reactions include gallic acid, sodium sulfite, cysteine, and tannins. Examples of nitrosamine levels in foodstuffs are summarized in Table 9.17. Cured meats (especially fried bacon), followed by fish and cheese, represent the major sources of nitrosamines in the diet. The formation of these derived toxicants in foods is also dependent on the condi-

Table 9.16 Nitrosamine Precursors That Contaminate Foodstuffs Compound Atrazine Benzthiazuram Carbaryl Fenuron Ferbam Morpholine Propoxur Simazine Succinic acid 2,2′-dimethyl hydrazide Thiram Ziram Source: From Concon (1988).


Chemical class

Nitrosamine derivative

Secondary amine Carbamate Carbamate Carbamate Amide Secondary amine Carbamate Secondary amine Amide Amide Amide

N-Nitrosoatrazine N-Nitrosobenzthiazuram N-Nitrosocarbaryl Dimethylnitrosamine Dimethylnitrosamine N-Nitrosomorpholine N-Nitrosopropoxur N-Nitrososimazine Dimethylnitrosamine Dimethylnitrosamine Dimethylnitrosamine

Table 9.17 Nitrosamine Levels in Foodstuffs Food Bacon, raw Bacon, fried

Bacon, frying fat drippings Luncheon meat Salami Danish pork chop Sausage Sausage, mettwurs Chinese Fish Sable, raw Salmon, raw Shad, raw Sable, smoked Salmon, smoked Salmon, sable, and shad, smoked and nitrate/nitrite cured Salted, marine fish Fish sauce Cheese Baby foods Shrimp, dried NPyr Shrimp sauce Squid

Canned meats Ham and other pork products Beef products Wheat flour

Nitrosaminea

Level, ppb

DMN, DEN, NPyr NPip NPyr DMN, NPyr NPyr

0 1–40 10–108 11–38 2–30 10–108

DMN, DEN DMN, DEN DMN, DEN DMN NPyr, NPip DMN

1–4 1–4 1–4 1–3 13–105 0–15

DMN DMN DMN DMN DMN DMN

DMN DMN, NPyr DMN DMN DMN 0–37 DMN NPyr DMN NPyr DMN DMN DMN DEN

4 0 0 4–9 0–5 4–26

50–300 0–2 1–4 1–3 2–10 0–10 0–10 2–8 0–7 1–3 0–5 1–2 0–10

a

DEN, diethylnitrosamine; DMN, dimethylnitrosamine; NPyr, nitrosopyrrolidine; NPip, nitrosopiperidine. Source: Modified from Concon (1988).

The biological activity of N-nitroso compounds has been studied extensively. The toxicity of nitrosamines was first recognized in 1937 by Freund, who reported two cases of accidental poisoning from inhalation of DMN. In 1956, Magee and Barnes reported that DMN was a potent hepatocarcinogen in rats. This report marked the beginning of worldwide interest in N-nitroso compound carcinogenesis. Since then, a large number of N-nitroso


compounds have been tested for carcinogenicity. Among the 400-odd N-nitroso compounds assayed thus far for carcinogenicity, over 90% have yielded positive findings. The most widely tested N-nitroso compound, N-nitrosodiethanolamine (NDEA), has been shown to be carcinogenic in 40 species (Lijinsky, 1987). There are important distinctions between nitrosamines and nitrosamides: the former must be activated to carcinogens by oxidative enzymes (e.g., cytochrome P-450), whereas the latter are direct-acting carcinogens. Nitrosamines often produce tumors at a site(s) distant from the point of application. Selected N-nitroso compounds that may be found in foods and their corresponding target organs are listed in Table 9.18. A number of these compounds seem to have a broad organotropicity in a single species. The organ specificity depends on the chemical nature of the N-nitroso compound and may depend on the dose, route of administration, and animal species. Similarly, there are large interspecies differences in the organs affected by the same compound. N-Nitroso compounds have the ability to induce transplacental carcinogenesis. In rats, kidney tumors have been induced transplacentally by DMN; similar tumors of the lung and liver have been induced in mice (Concon, 1988). The effect observed in the offspring depends on the time during gestation when the N-nitroso compound is administered. Generally, embryotoxic effects are observed when the administration is on days 1–10, teratogenic effects on days 9–16, and carcinogenic effects from day 10 to delivery (Archer, 1982). Several factors influence the carcinogenicity of Nnitroso compounds. Potentiating factors include hormones and their levels, other carcinogens or toxicants, viral or bacterial infections, metals, and nutritional factors. Synergism is often observed in the presence of mycotoxins. In contrast, reducing agents such as ascorbic acid, cysteine, and tannins diminish the carcinogenic potential of N-nitroso compounds. No animal species tested thus far is resistant to DMN or DEN, the two nitrosamines commonly found in foodstuffs (Lijinsky and Taylor, 1977). Thus, the common consensus is that humans cannot be expected to be resistant to the nitrosamines (Mirvish, 1977, 1983). These compounds are particularly effective when exposure is through the oral route, at small doses, and over a long period, conditions particularly relevant to humans. Furthermore, these compounds are systematically organotropic and induce tumors in target tissues independently of the route of administration. Biochemical studies with human liver in vitro have produced evidence that nitrosamines are metabolized and interact with nucleic acids (Montesano and Magee, 1974). This finding suggests that human me-

Table 9.18 Target Tissues of Selected Nitrosamines Found in Foods Nitrosamines

Target tissues

Dimethylnitrosamine

Liver

Diethylnitrosamine

Kidney Lung Nasal cavities Liver

Di-N-propylnitrosamine

Di-N-butylnitrosamine

N-Nitrososarcosine N-Nitrosopyrrolidine

N-Nitrosopiperidine

N-Nitrosomorpholine

N-Methyl-N-nitrosourea

Kidney Lung Nasal cavities Esophagus Forestomach Larynx Trachea Bronchi Liver Esophagus Tongue Liver Lung Esophagus Bladder Forestomach Trachea Tongue Esophagus Liver Lung Nasal cavities Trachea Testes Liver Lung Nasal cavities Esophagus Larynx Trachea Testis Liver Lung Kidney, nasal cavities, ovaries, esophagus Trachea, larynx, bronchus Central nervous system Peripheral nervous system Intestines Kidney Forestomach Skin

Test species Rat, mouse, European hamster, guinea pig, rabbit, rainbow trout, newt, mink, mastomys (Praomys natalensis), aquarium fish (Lebistes reticulates) Rat, Syrian golden and European hamsters Syrian golden hamster Rat, rabbit Rat, mouse, Syrian golden hamster, Chinese hamster, guinea pig, rabbit, dog, pig, trout, grass parakeet, monkey, Brachydanio rerio Rat Mouse, Syrian golden hamster Rat, mouse, Syrian golden hamster, European hamster Rat, mouse, Chinese hamster Mouse, Chinese hamster Syrian golden and European hamsters Syrian golden and European hamsters European hamster Rat Rat Rat Rat, mouse, guinea pig Syrian golden and Chinese hamsters Rat, mouse Rat, mouse, Syrian golden and Chinese hamsters, guinea pig Mouse, Syrian golden hamster, guinea pig Syrian golden hamster Mouse Rat Rat Mouse, Syrian golden hamster Rat Syrian golden hamster Rat Rat, mouse, monkey, Syrian golden hamster Mouse, Syrian golden hamster Rat Rat, mouse Rat, Syrian golden hamster Rat, Syrian golden hamster Mouse Rat, mouse Mouse Rat Syrian golden hamster Rat, mouse, rabbit, dog Rat, dog Rat, Syrian golden hamster, rabbit Rat, mouse Rat, mouse Rat, mouse, dog (table continues)


Table 9.18 (continued) Nitrosamines

Target tissues

Test species

N-Methyl-N-nitrosourea (continued)

N-Ethylnitrosourea

N-Methyl-N-nitrosourethane

N-Ethy-N-nitrosourethane N-Methyl-N′-nitro-Nnitrosoguanidinea

Subcutaneous tissues Glandular stomach, jaw, bladder, uterus, vagina Liver, lung, hematopoietic system Pharynx, esophagus, trachea, bronchus, oral cavity Stomach, pancreas, ear duct Central nervous system Peripheral nervous system Kidney Hematopoietic system Skin, intestines, ovary, uterus Lung Forestomach Esophagus Kidney, intestines, ovary Pancreas, subcutaneous tissues Forestomach, intestines Glandular stomach

Syrian golden and European hamsters Rat

Guinea pig Rat, mouse Rat, mouse Rat, mouse Rat, mouse Rat Rat, mouse Rat, mouse, Syrian golden hamster Rat, Syrian golden hamster Rat Rabbit Rat Rat, Syrian golden hamster

Forestomach Stomach Intestines Skin Subcutaneous tissues Lung

Rat, mouse Dog Rat, mouse, Syrian golden hamster, dog Mouse Rat Rabbit

Mouse Syrian golden hamster

a

Not found in foods as such, but one of similar structures may be derived from naturally occurring guanidines, such as methylguanidines. The latter has been detected in certain foods, e.g., meats, and is probably derived from creatinine. Source: From Magee et al. (1976) and Concon (1988).

tabolism of nitrosamines may also produce proximate carcinogens similar to those seen in almost every animal study. When the amounts of total nitrosamines in food, water, and other sources are added to those formed throughout the GI tract, the total nitrosamine load of modern populations would be considerable. Viewed from this perspective, each carcinogen, even in trace amounts, assumes considerable significance.

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10 Toxicants and Antinutrients in Plant Foods

10.1 INTRODUCTION Animal life on earth is primarily sustained by green plants with photosynthetic capacity to convert carbon dioxide and water into basic macronutrients, i.e., carbohydrates, protein, and fat. In fact, on a global basis over 65% of food protein and over 80% of food energy is supplied by plants, and in terms of gross tonnage, plant products directly contribute about 82% of the total world food harvest (Deshpande, 1992). The photosynthetic process of plants, however, is not confined to the production of basic macronutrients. It also includes the biosynthesis of a variety of organic compounds. Traditionally, the processes generating plant compounds have been categorized as either primary or secondary metabolism. Research in plant physiological characteristics since the 1980s, however, has clearly shown that such a distinction between primary and secondary metabolites is at best arbitrary. The once-popular view of a secondary metabolite as one that does not play an indispensable role in plant life at the cellular level is no longer valid. It is now widely recognized that plants do not haphazardly produce a large number of chemical compounds; rather, each metabolite is biosynthesized for a definite purpose, and all products are interrelated according to a complex process that conserves energy and scarce organic nutrients. Antinutritional or toxic compounds that occur naturally in many plants can be considered secondary metabolites. Most secondary metabolites are now known to be essential to plant life; many of them provide a defense mechanism against bacterial, viral, and fungal attack analogous to the immune system of animals. Many are also produced in large amounts as a direct result of some adverse environmental condition.


The science of nutrition is not just the science of food and its relation to life and health. We must be concerned not only with what is required in a diet, but also with what is actually consumed. We should be as concerned with the problems caused by an excess of a food component as we are with the problems caused by the deficiency of an essential nutrient. In considering the physiological effects of food components, it should be noted that these effects are always related to the level of their intake. A useful concept is that for every food component, there are three ranges of intake: one associated with physiological inertness, a second with physiological function or benefit, and the third with potential hazard. Although it is arguable at what level a nutrient is physiologically inert, there is no doubt that certain levels of intake are insufficient to maintain normal body functions. The level of nutrient requirement associated with normal health, i.e., with physiological function and benefit, is well understood for most nutrients. We also know with certainty that the concept of “zero risk” cannot be considered valid anymore. One would probably not consider our food sources of energy as ever constituting a potential hazard, but there is a consensus among nutritionists that currently the most important problem of malnutrition in the United States and in many developed Western countries is obesity. This example clearly shows that the margin between the level of caloric intake consistent with normal physiological function and benefit and that creating a potential hazard is narrow. Thus, for every nutrient there is also a level of intake that constitutes a potential hazard. The margin between the level of function and the level of hazard varies considerably with each and must be determined in each case.

That the basic components (i.e., carbohydrates, protein, and fat) of human diet under normal conditions do not exert any adverse effects is taken for granted. Natural foods in everyday diets also contain a great number of other potentially toxic substances. However, this does not necessarily mean that the food is hazardous to human beings. A substance that is considered toxic has a more or less pronounced capacity to induce deleterious effects on the organism when tested by itself in certain doses. However, this capacity is not always realized under usual dietary conditions. Humans consume a multitude of toxic substances in their normal diet every day without showing any signs of intoxification. This is probably because natural toxicants usually exert their effects only when they are consumed under special conditions or when there are other potentiating substances available. In addition, the concentration of toxicants occurring naturally in the food is often so low that the item must be consumed in usually unrealistic amounts every day for an extended period to allow intoxification to occur. Furthermore, it should be noted that humans can handle small amounts of various toxicants. Similarly, most toxic effects of various chemicals that are potentially hazardous do not have an additive effect. There also seem to occur antagonistic reactions that make some ingredients interfere with and reduce the toxic effects of other components. All these facts prompt Liener (1989) to prefer the term antinutritional to toxicants to denote such hazardous food components, since the former is not very restrictive and may be liberally interpreted to mean nothing more nor less than an adverse physiological response produced in humans. Antinutritional and toxic factors that commonly are present in the human food chain can be classified into two broad groups: those that occur naturally (natural or inherent) as a result of intrinsic metabolism of the animal or plant and those that are formed (acquired) as a result of microbial growth, accumulated from the environment, or unintentionally introduced during handling, processing, and storage. In this chapter, only the naturally occurring antinutritional and toxic factors of important plant food sources are discussed. Because of the obvious limitations of space, coupled with the fact that several excellent reviews and books on various aspects of naturally occurring food toxicants are available, no attempt has been made to cover all of the natural toxic substances known to be present in plant materials. Therefore, only certain evolutionary, structural, biochemical, technological, nutritional, and toxicological aspects of most important antinutritional factors that occur in the human food chain are discussed.


10.2 PROTEINASE (PROTEASE) INHIBITORS Protein inhibitors of proteinases (or protease inhibitors) are ubiquitous. They are present in multiple forms in numerous tissues of animals and plants as well as in microorganisms. Their gross physiological function is the prevention of undesirable proteolysis, but detailed physiological functions have been only rarely elucidated. These inhibitors have attracted the attention of scientists in many disciplines. Nutritionists are concerned with their possible adverse effects on the nutritive value of plant proteins. The inhibitor-enzyme reactions have provided a simple model system for protein scientists to study protein-protein interactions as well as enzyme mechanisms. Because of their unique pharmacological properties, these inhibitors hold considerable promise in clinical applications in the field of medicine. Proteinase inhibitors were initially classified on the basis of protease inhibited, such as trypsin inhibitor. They can also be grouped on the basis of the class of protease inhibitor (Table 10.1). More recently, classification has also been based on similarities in the primary amino acid sequence and/or the disulfide bond location (Whitaker, 1997). Although the inhibition of proteolytic enzymes by extracts from animal tissues was first demonstrated in the 19th century (Fredericq, 1878), it was only in the 1930s that their presence in plant material was recognized. Read and Haas (1938) reported that an aqueous extract of soybean flour inhibited the ability of trypsin to liquefy gelatin. This report was soon followed by the first isolation of a

Table 10.1 Families of Plant Proteinase Inhibitors Serine protease inhibitors (serfins) Bowman-Birk (trypsin/chymotrypsin)a Kunitz (trypsin, others)a Potato I (chymotrypsin, trypsin)a Potato II (trypsin, chymotrypsin)a Cucurbit (trypsin) Cereal superfamily (amylase, trypsin)b Ragi I-2 family (amylase, protease)b Maize 22 kDa/thaumatin/PR (amylase, trypsin)b Cysteine protease inhibitors (cystatins and stefins) Cystatin superfamily Cystatin family Stefin family Fitocystatin family Metalloprotease inhibitors Carboxypeptidase a

Second enzyme listed binds less tightly. Double-headed inhibitor.

b

plant proteinase inhibitor from soybeans by Kunitz (1945, 1946). A year later, the first systematic study of plant proteinase inhibitors was made, by Borchers and Ackerson (1947). Because of the great and increasing importance of grain legumes as food and feed, research during the next period mainly concentrated on seeds from the legume family. A summary of our current knowledge of the distribution of these inhibitors in different food legumes is shown in Table 10.2. The proteinase inhibitors are nonglycosylated, water-soluble (albumin) proteins that account for about 0.2% to 2% of the total soluble protein of the legume seeds (Sgarbieri and Whitaker, 1982; Deshpande and Damodaran, 1990). Most are low-molecular-weight (4000- to 8000-Da) proteins. Two major families of proteinase inhibitors have been described in legumes: the BowmanBirk–type and the Kunitz-type inhibitors. They are distinct families of proteins, as evidenced by their molecular weights, compositions, and amino acid sequences. The im-

Table 10.2 Distribution of Protease Inhibitors in Legumes Species

Protease inhibitorsa

Arachis hypogaea Cajanus cajan Canavalia ensiformis Cicer arietinum Cyamopsis tetragonoloba Dolichos biflorus Dolichos lablab Glycine max Lathyrus odoratus Lathyrus sativus Lens esculenta Lupinus albus Phaseolus aconitifolius Phaseolus angularis Phaseolus aureus Phaseolus coccineus Phaseolus lunatus Phaseolus mungo Phaseolus vulgaris Pisum sativum Psophocarpus tetragonolobus Vicia faba Vigna unguiculata Voandzeia subterranea

T, C, Pl, K T T, C, S T, C T, C, S T T, C, Th T, C T T, C T T T T, C T, endopeptidases T, C T, C T, C, S T, C, E, S T T T, C, Th, Pr, Pa T, C T

a

C, chymotrypsin; E, elastase; K, kallikrein; Pa, papain; Pl, plasmin; Pr, pronase; S, subtilisin; T, trypsin; Th, thrombin. Source: Compiled from Liener and Kakade (1980) and Deshpande and Sathe (1991).


portant properties of these two inhibitor classes are described in the following discussion. 10.2.1

Bowman-Birk Inhibitors

The Bowman-Birk inhibitor, also referred to as the acetone-insoluble inhibitor, was first recognized by Bowman (1944) and subsequently purified and characterized by Birk and coworkers (Birk, 1961; Birk et al., 1963). It is found in the seeds of all common agriculturally important legume species. These trypsin inhibitors generally contain between 60 and 85 amino acid residues in a single polypeptide chain (Norioka and Ikenaka, 1983), yielding a molecular weight of approximately 8000 Da. However, many members of this family exhibit strong self-association in solution and thus often appear considerably larger (usually dimer or trimer) in the absence of denaturing agents, such as urea (Birk, 1985). The amino acid composition of the Bowman-Birk inhibitors indicates a very high content of Cys (14 residues), all involved in disulfide bonds (Mossor et al., 1984). The inhibitors also have a relatively high content of Asp, Asn, and Ser. Met, Val, Tyr, and Phe are found in small quantities; Trp is generally absent. Because of their low molecular weight and a high content of disulfide bonds, Bowman-Birk inhibitors are generally considered to be heat stable (Mossor et al., 1984). The primary structures of Bowman-Birk trypsin inhibitors from various sources are invariably similar (Wilson, 1981; Norioka and Ikenaka, 1983). They are double-headed, i.e., generally capable of simultaneously and independently inhibiting two molecules of proteases (Laskowski and Kato, 1980). The two enzyme molecules may be the same (e.g., trypsin) or may be different (e.g., one trypsin and one chymotrypsin or elastase). This property is also reflected in their amino acid sequence. A high degree of homology is observed between the first half of the sequence, which contains the reactive site for one proteinase molecule, and the second half of the sequence, which contains the second reactive site. This internal homology suggests the evolution of the present-day doubleheaded inhibitors from an ancestral single-headed inhibitor by a partial gene duplication event (Wilson, 1981). 10.2.2

Kunitz Inhibitors

The Kunitz-type proteinase inhibitors typically contain 170 to 200 amino acid residues, with a molecular weight of about 20,000 Da (Liener, 1983). In soybeans, the inhibitor is a single polypeptide chain. They are single-headed, inhibiting one molecule of the enzyme (generally trypsin

or chymotrypsin) per molecule of the inhibitor. Although the Kunitz-type inhibitors are absent from many agriculturally important members of the legumes, such as Phaseolus, Pisum, and Vigna species, they are found in soybeans and winged beans. Since all naturally occurring trypsin inhibitors have bonds Lys-X or Arg-X in the cavity of their structure as a result of S-S bonds (Laskowski and Kato, 1980), they can also be classified according to the requirements for either Lys or Arg at their reactive sites. Thus, Kunitz inhibitors can be classified as arginine inhibitors, whereas BowmanBirk inhibitors are of the lysine type. The trypsin inhibitors that have Arg at the reactive site lose activity on modification of Arg, but not of Lys. Conversely, Lys-type inhibitors are still active on modification of Arg residues. The compositional and structural aspects of proteinase inhibitors, their physiological significance, as well as the mechanisms of interactions with various proteinases have been discussed in several excellent reviews (Laskowski and Kato, 1980; Liener, 1983; Sgarbieri and Whitaker, 1982; Liener and Kakade, 1980; Weder, 1986). The available literature data on proteinase inhibitors from various legumes are summarized in Table 10.3. Most

inhibitors show inhibiting effects against trypsin and chymotrypsin, but some also inhibit other enzymes, such as elastases, papain, plasmins, and thrombins. Also, in some species, isoinhibitors are frequently found. Examination of the compilation in Table 10.4 reveals that most legume species contain less than 50% of the trypsin inhibitory activity (TIA) of soybeans. Particularly low activities are present in most cultivars of Vicia, Pisum, and Lupinus species and a few cultivars of Phaseolus vulgaris. Legumes with at least 60% to 75% of the TIA in soybeans include Cajanus sp., Phaseolus lunatus, and Cicer sp. (Soni et al., 1978; Rackis et al., 1986). Although trypsin inhibitors from food legumes, particularly soybeans, are studied most widely, they are also found in several other food products, including the staple cereals and various meat products (Table 10.5). However, compared with legumes, these foods have attracted rather limited attention in this respect. The fact that proteinase inhibitors are so widely distributed among those very plants that constitute an important source of dietary protein throughout the world has stimulated a vast amount of research into their possible nutritional significance. Because of the important role of soy-

Table 10.3 Characteristics of Proteinase Inhibitors in Food Legumes MW, daltons

Legume Glycine max (soybean)

Phaseolus lunatus (lima bean) Phaseolus vulgaris (Great Northern) Phaseolus vulgaris (navy bean) Phaseolus vulgaris (pinto bean) Phaseolus aureus (mung bean) Vicia faba (broad bean) Cicer arietinum (chickpea) Vigna unguiculata (cowpeas)

a

Specificitya

21,700

T

8,000

T, C

9,000

T, C

8,086 I, 8,371 II, 8,884 IIIb 23,000 7,900 19,000

T, C

12,000

T, C T, C

11,000

T, endopeptidase of mung bean T, C

10,000

T, C

8,000

T

8,000

T, C

T, trypsin; C, chymotrypsin.


Comments Has Trp, primary sequence of 181 amino acids is known, single-headed Kunitz type Double-headed Bowman-Birk type, contains seven disulfide bonds Double-headed inhibitor, has at least four to six isoinhibitors Chymotrypsin weakly inhibited by 1 and II, and strongly by IIIb, which has independent sites for the two enzymes Four possible isomers present Two isoinhibitors present, possibly different binding sites for trypsin and chymotrypsin Another inhibitor of MW 2000 also reported Also inhibited thrombin, pronase, and papain (slightly) Independent binding sites for trypsin and chymotrypsin, two to four isoinhibitors present as a result of proteolysis Binds with two moles of trypsin simultaneously Independent binding sites for trypsin and chymotrypsin

Reference Odani and Ikenaka (1972) Odani and Ikenaka (1972) Stevens et al. (1974) Birk (1976)

Whitley and Bowman (1975) Wang (1975) Beumgartner and Chrispeels (1976) Warsy et al. (1974) Belew et al. (1975), Smirnoff et al. (1979) Gennis and Cantor (1976) Gennis and Cantor (1976)

Table 10.4 Trypsin Inhibitory Activity Content of Selected Food Legumes in Relation to Soybeans

Botanical name Cajanus cajan Cicer arietinum Lens esculenta Lupinus spp. Phaseolus aconitifolius Phaseolus aureus Phaseolus lunatus Phaseolus mungo Phaseolus vulgaris Pisum sativum Vicia faba Vigna unguiculata a

Common name

Relative TIA (soy = 100%)a

Reference

Pigeon pea Chickpea Lentil Lupin Moth bean Green gram, mung Lima bean Black gram Dry beans Pea Fava/broad bean Cowpea

60 66 25 0 27 37 77 52 13–44 1.5–13 0.7–36 11.1–28

Soni et al. (1978) Soni et al. (1978) Soni et al. (1978) Valdebouze (1977) Soni et al. (1978) Soni et al. (1978) Hove and King (1979) Soni et al. (1978) Hove and King (1979) Valdebouz (1977) Valdebouz (1977) Valdebouz (1977)

TIA, trypsin inhibitory activity.

beans in animal feeding and their potential contribution to human nutrition, inhibitors from this particular legume have received special attention. In this regard, the effect of raw soybeans on growth has been extensively studied. Earlier reports found that the slight increase in apparent protein digestibility on feeding heated soybean meal to animals was too small to account for the pronounced improvement in their growth rate (Melnick et al., 1946; Mitchell et al., 1945). These studies on protein digestibility, however, were based on measuring fecal nitrogen excretion. Later on, Carroll and associates (1952) studied soy protein digestibility by measuring net protein absorption in

Table 10.5 Nonlegume Foods Containing Proteinase Inhibitor Activity Cereals Rice, wheat, corn, triticale, rye, barley, millet, sorghum, buckwheat, cereal-based products Roots and tubers Potato, sweet potato, yam, taro, cassava Vegetables Cabbage, tomato, lettuce, radish, onion, carrot, sweet corn Fruits and nuts Brazil nut, apple, banana, orange, raisin Animal foods Milk, cheese, egg, beef, lamb, pork, poultry, sweetbread, fish, meat-based products Miscellaneous Table sugar, butter, margarine, tea, coffee, drinking chocolate, milk chocolate Source: Compiled from Doell et al. (1981) and Rackis et al. (1986).


rats. They observed that animals fed raw soy meal absorbed less than half the nitrogen of those fed heated meal. These studies suggested that feeding raw legumes results in a considerable loss of protein from the site of its absorption in the small intestine. Consequently, although fecal nitrogen is only slightly increased in animals fed raw vs. heated soybeans, less nitrogen is effectively utilized. The discovery of a heat-labile trypsin inhibitor suggested a hypothesis for this growth-depressing effect of soybeans. The inhibitor can interfere with protein digestion by complexing with trypsin and thus preventing the proteolysis of both endogenously secreted and dietary proteins. The fact that raw soybeans or the trypsin inhibitor itself could cause pancreatic hypertrophy and hyperplasia, an effect that is accompanied by an increase in the secretory activity of the pancreas (Liener, 1983), has ultimately led to a better understanding of the mode of action of the soybean trypsin inhibitor. The enzyme-inhibitor complex is neither degraded nor reabsorbed. Since pancreatic proteases are particularly rich in S-amino acids, this loss of essential amino acids from endogenous sources, in addition to the incomplete proteolysis of dietary proteins, contributes to the soy-induced growth depression. This relationship is further supported by the fact that supplementing raw soy meal with S-amino acids improves the growth rate to that of the heated meal (Borchers, 1961). Although part of this improvement undoubtedly results because soy meal itself is limiting in these amino acids, the growth response with their supplementation is much greater in animals fed a raw soy flour diet than the heated flour (Gertler et al., 1967). This finding is consistent with an increased loss of S-rich endogenous proteins due to

trypsin inhibitors. Thus, increasing the dietary supply of amino acids in terms of either protein quality or protein quantity supports better growth by compensating for endogenous losses. The mechanism whereby the trypsin inhibitor causes pancreatic hypertrophy and increases proteolytic enzyme concentrations may be explained in terms of the mechanism of the regulation of pancreatic secretion. Lyman and coworkers have shown that the levels of free intestinal trypsin and chymotrypsin monitor the secretion rate by a negative feedback inhibition involving a hormone, chloecystokinin-pancreozymin (Green and Lyman, 1972; Lyman et al., 1974; Schneeman and Lyman, 1975). This negative feedback mechanism is altered when trypsin is complexed with the inhibitor. This, in turn, releases the hormone, thus increasing the pancreatic juice flow. Although trypsin inhibitors contribute to the growth depression in animals fed raw legumes, other factors may also be involved. Kakade and colleagues (1973) observed that when the inhibitor-free raw soy meal was fed to rats, the degree of growth depression was only marginally less than that of the raw meal containing the inhibitor. This suggested that much, if not most, of the growth depression effect is due to the raw soybean protein itself. Studies in the late 1980s clearly showed that the native storage proteins of various food legumes are resistant to proteolysis (Deshpande and Nielsen, 1987; Nielsen et al., 1988; Deshpande and Damodaran, 1989a, 1989b). The prolonged presence in the intestine of protein of poor digestibility may act by a mechanism similar to that of trypsin inhibitors. It is now well established that most raw or partially cooked legumes are of low nutritional value, an observation that is attributed to the presence of the inhibitors as well as lectins. Fortunately, not only are the inhibitors found in low amounts in most legumes, but most of them are quite heat-labile. Hence, proteinase inhibitors pose few, if any, serious nutritional problems in properly processed legumes. The significance of proteinase inhibitors in human and animal nutrition has been comprehensively reviewed by several authors. The consensus is as follows: (a) Inhibitors of trypsin stimulate the biosynthesis of the enzymes of the pancreas, causing increased requirements of the necessary amino acids in animals. This leads to an increase in the transformation of Met to Cys in the pancreas. The increased requirement of S-amino acids, coupled with their deficiency in legume proteins, cannot be adequately compensated for by the dietary proteins. The enhanced secretion of pancreatic enzymes results in pancreatic hypertrophy in laboratory animals fed legumebased diets. (b) The trypsin inhibitors decrease the proteolysis of dietary proteins by forming trypsin-inhibitor com-


plexes. These complexes are not broken down even in the presence of adequate amounts of enzymes. This effect further depresses animal growth, since such undigested complexes cannot be assimilated and hence excreted. Since the proteinase inhibitors are rich in Cys, this characteristic further lowers the availability of S-amino acids. Although there is little doubt that the proteinase inhibitors can produce adverse physiological effects in animals, the question naturally arises as to whether they have any physiological significance to humans. Their practical significance with respect to human nutrition is still speculative. TIA is invariably measured in vitro by its ability to inhibit bovine or porcine pancreatic trypsin, which is commercially available. The human trypsin exists in both a cationic form, which is the major component of human pancreatic juice, and an anionic form, which constitutes about 10% to 20% of the total trypsin activity (Weder, 1986). Whereas the less active anionic trypsin is fully inactivated by the soybean trypsin inhibitor, the predominant cationic species is only weakly inhibited (Figarella et al., 1974). That there is a causal relationship between the extent of in vitro trypsin inhibition and pancreatic hypertrophy in vivo has been largely assumed. The practical insignificance of trypsin inhibitors in humans as compared to that in rats was further elaborated by Liener (1977). He discussed the relationship between the size of the pancreas in various animal species and their susceptibility to pancreatic hypertrophy induced by raw soybean meal and trypsin inhibitors. Only animals whose pancreas weighed greater than 0.3% of their total body weight exhibited hypertrophy when fed raw soybeans. In humans, the size of the pancreas is only 0.09% to 0.12% of body weight. If the pancreatic size reflects its true functional activity, as suggested by Goss (1966), then the physiological response to raw soybeans or the inhibitors would likely differ in different animal species. Thus, it seems logical to conclude that despite the considerable body of evidence that implicates the proteinase inhibitors as contributory factors in the poor nutritive value of raw legumes in animals, their relevance to human nutrition remains uncertain. Deshpande (1992) critically reviewed the significance of trypsin inhibitors in human nutrition. Summarizing the various studies conducted during the 1981–1990 period (Table 10.6), he concluded that the inhibitor activity can readily be destroyed by more than 90% if the legumes are processed properly, i.e., by at least 30- to 60minute boiling water treatment or 15- to 20-minute autoclaving at 15 psi. The residual inhibitor activity of less than 10% may be attributable to nonspecific inhibition of trypsin by other dietary components such as phytates, tannins, and crude fiber as well as to our inability by the currently available techniques to detect low levels of inhi-

Table 10.6 Studies Reporting Effects of Processing on Proteinase Inhibitor Activities of Food Legumesa Country of origin

Legume

United States

Vigna unguiculata

India United States

Cicer arietinum, 8 desi and 7 kabuli types Arachis hypogaea

United States

Vigna unguiculata

United States United States

Glycine max Phseolus vulgaris, two cultivars

India

V. aconitifolia

Japan

Psophocarpus tetragonolobus

India United States

Cyamopsis tetragonoloba, four cultivars G. max, two cultivars

Japan

G. max

United States

P. vulgaris

United States/Japan United States

Lupinus angustifolius P. vulgaris, two cultivars

United States

P. tetragonolobus

Guatemala

P. vulgaris, three cultivars

Iraq England

V. faba, C. arietinum, V. unguiculata, V. radiata, and Lens culinaris V. faba, Pisum spp.

England

P. tetragonolobus

Sri Lanka United States

Mucuna utilis G. max

a

Inferenceb Over 92% TIA destroyed within 12 min of conventional heating (boiling in water) and 6 min microwaving Both TIA and CIA destroyed when heated for 60 min (only one time interval used) TIA completely destroyed by heat treatment for 180 min at 100°C or 45 min at 120°C At 100°C, heating cowpea flours of 19.4% and 25.5% initial moisture content, TIA decreased by over 90% within 6 and 1.75 min, respectively Autoclaving at 121°C for 30 min destroyed 98.9% TIA of soymeal Cooking for 35 min or frying at 180°C for 6 min inactivated TIA effectively, only75% CIA destroyed By ordinary cooking, pressure cooking after soaking in plain water or salt solution, and ordinary cooking of sprouts TIA “almost” eliminated from moth beans TIA in dry whole winged bean seeds completely inactivated after microwave heating for 5 min By autoclaving (1 kg/cm2, time not mentioned) of meals over 98% TIA destroyed By steaming or cooking for 20 min “as commonly practiced” TIA completely eliminated in immature seeds, soaking (24 hr) followed by cooking (20 min) essential for mature seeds TIA in soybeans of 24.3% moisture soaked for 1 hr inactivated completely after microwave treatment (2450 MHz) for 4 min, 6 min for unsoaked soybeans In conventionally cooked beans (soaked in water and boiled), loss of 97.2% TIA TIA completely lost on heating at 90°C for 8 min By canning of navy beans by two different methods >95% TIA destroyed: open-kettle cooking at 100°C for 10 min 92% TIA; TIA completely eliminated after 40 min cooking at 100°C Winged bean tuber TIA and CIA easily inactivated by wet heat after 2-min heating in boiling water By cooking of black, white, and red beans antitryptic activity eliminated, residual activity attributed to tannins to a large extent By heating of soaked seeds at 121°C for 30 min completely TIA in all destroyed By autoclaving (170 kPa for 10 min) TIA and CIA in both species completely destroyed; little effect of dry heat By infrared treatment, autoclaving, and boiling water treatments over 95% TIA destroyed; no effect of microwave (15% moisture meal, 10 min) and dry heat (100°C for 60 min) By cooking TIA completely destroyed In autoclaved soy flakes >90% TIA destroyed

Representative studies from the 62 research papers published during the 1981–90 period in the three major food science journals (J Food Sci, J Agric Food Chem, and J Sci Food Agric). These were selected to represent different food legume species and diverse processing conditions b TIA, trypsin inhibitor activity; CIA, chymotrypsin inhibitor activity. Source: Deshpande (1992)


bition. Generally, depending on the product desired, legumes are often cooked for 1 to 3 hours at atmospheric pressure. These cooking procedures should therefore effectively destroy the proteinase inhibitor activities of various food legumes. Moreover, legumes are often preprocessed by one of several different methods (soaking overnight is not uncommon) that alone may reduce the inhibitor activity to varying degrees. The possibility of residual inhibitor activity that would be nutritionally detrimental in thus-processed legumes therefore appears to be quite small. Unfortunately comprehensive studies concerning the actual dietary intake of trypsin inhibitors from various foods in the daily human diet are lacking. One such rare study estimated that the average British diet provides 330 mg TIA per person per day (Doell et al. 1981). In this study, diets comprising substantial amounts of egg, potato, brown bread, and some vegetables contained more TIA than those based on white bread and rice (polished?). Similarly, of the total of 294.6 mg of actual TIA ingested daily from the British household diet, eggs (93.5 mg), milk and milk products (56.8 mg), and potatoes (42.5 mg) alone contributed over 65% of the daily intake (Table 10.7). In contrast, the Asian soy-based foods included in this particular study contained only a marginal degree of residual TIA. This finding is in striking contrast to the food habits in Third World countries, where the per capita consumption of animal foods such as eggs and milk products is rather limited as a result of either nonavailability or various socioeconomic factors and religious beliefs. To illustrate the point further, let us assume that TIA accounts for 2% of total legume protein (Liener, 1976) and that legumes contain, on an average, 25% protein. If 90%

Table 10.7 Average Daily Protein and Trypsin Inhibitor Intake from the British Household Diet

Food group Milk and milk products Meat Fish Eggs Vegetables Potatoes Other Fruits and nuts Bread and cereals Other foods Total

Protein, g/person/day

Trypsin inhibitor, mg/person/day

17.9 19.5 2.9 3.7

56.8 19.8 1.5 93.6

2.8 3.8 0.7 18.7 1.0 71.3

42.5 37.8 11.8 25.3 5.5 294.6

Source: Doell et al. (1981).


TIA is destroyed during cooking (the data presented in Table 10.6 certainly indicate so), an average serving of 100 g beans on a dry weight basis contributes 50 mg (100 mg × 0.25 × 0.02 × 0.1) residual TIA to daily dietary intake. To account for the 193-mg TIA intake in the average British household diet contributed by just the three food groups mentioned, one then needs to consume approximately 386 g beans (equivalent to almost 100 g protein per person per day, rather unrealistic statistics given the per capita legume consumption) on a dry weight basis. Because beans contain 65% moisture (as is, based on 1.5-g water uptake per 1 g of beans with optimal cooking [Deshpande and Cheryan, 1986]) on cooking, this amounts to a staggering portion of over 950 g cooked beans per person per day. Even if we follow the rather unrealistic conservative approach that only 50% of the original TIA is destroyed, this still amounts to approximately 77 g beans (or about 200 g cooked beans) per person per day. Globally, only the per capita consumption of legumes of certain population segments in India and a few selected African countries would rival these figures. The survey of Doell and coworkers (1981) raises yet another serious question. Free fatty acids, especially linoleic acid, appeared to inhibit trypsin in their study. Both fat and protein breakdown occur predominantly in the intestinal region. On a diet rich in fat, the likelihood of trypsin inhibition by fatty acids therefore should be of equal importance to that of proteinase inhibitors of legumes. Under such conditions, do the fatty acids stimulate endogenous trypsin secretion, thereby predisposing humans to pancreatic hypertrophy, as is often reported in laboratory animals fed “raw” legume flours (typically soy) or purified trypsin inhibitor from legumes, and is the rationale often speculated to have significance to human nutrition? The negative feedback control mechanism whereby the secretory activity of the pancreas is subject to control by the level of trypsin in the intestinal tract should then also be equally effective for a fat-rich diet. Deshpande (1992) further discussed the normal and worst-case scenarios of dietary intake of TIA from legumes from typical U.S. and Indian diets (Table 10.8). The average dry bean consumption in the United States has remained steady at about 8.5 g per person per day since the 1930s. A typical Indian diet generally contains about 50 g beans per person per day, although the average intake ranges from 35 to 135 g, depending on the population segment (Deshpande and Deshpande, 1991). Even if the per capita consumption of beans were to be doubled in the coming few years (the RDA guidelines do suggest an increased intake of legumes in U.S. diets), the dietary intake of residual TIA from properly processed legume-based foods should not exceed 0.01 g/day and 0.025 g/day in the

Table 10.8 Normal and Worse-Case Scenarios of Contribution of Proteinase Inhibitors from Legumes in the Human Diet Consumption, g/day/person United States Parameter a

Protein intake from legumes , g Contribution of proteinase inhibitors to dietb, g Actual intake if 50% destroyed during processing, g Actual intake if 90% destroyed during processing, g Probable loss of activity during gastric digestion in presence of other dietary constituents Extent of breakdown due to pepsin and other nonspecific proteinases during intestinal digestion Extent of residual inhibitor complexed to other dietary constituents, not digested and therefore excreted Probability of complexation with trypsin and chymotrypsin during intestinal transit time (maximum 2–3 hr, ?) of ingested food Rate and kinetics of complexation with enzymes under less than ideal conditions of other dietary components present at the same time Probability of human adaptation to presence of residual inhibitor in daily diet during minimum 7000-year history of legume domestication Pancreatic hypertrophy in humans Probability of deaths

India

10

20

50

100

150

2.5 0.05 0.025 0.005 ?

5.0 0.10 0.05 0.01 ?

12.5 0.25 0.125 0.025 ?

25 0.50 0.25 0.05 ?

37.5 0.75 0.375 0.075 ?

?

?

?

?

?

?

?

?

?

?

?

?

?

?

?

?

?

?

?

?

Likely to be very high

??

?? ?? ?? Extremely unlikely

??

a

Assuming an average of 25% protein in food legumes. Based on proteinase inhibitors’ constituting, on an average, 2% of the total legume protein. Source: Deshpande (1992). b

United States and India, respectively, under normal circumstances. We then enter a completely gray area of their relevance to human nutrition as several questions remain to be answered (Table 10.8). The presence of other dietary constituents may accelerate gastric breakdown of these inhibitors; complexation with other proteins and minerals may change their potency, or such complexes may even be excreted. The kinetics of inhibitor binding with trypsin are altered in the presence of other dietary constituents compared with those observed under the ideal in vitro conditions in the laboratory. Similarly, legumes are high-fiber foods and thus have shorter residence times in the intestine than predominantly cereal- or meat-based diets (Hellendoorn, 1979). If these factors even marginally influenced the binding of trypsin to the residual inhibitor, the actual residual levels of TIA would be even lower and rather inconsequential. Deshpande (1992) considered yet another scenario in this respect. Soy protein products have long been an ac-


cepted ingredient in various processed food products in the United States. What would be the dietary TIA intake from such adequately processed soy products? The average daily intake of soy products from various processed foods in Japan is estimated to be 9.5 g per person per day (Evans, 1980; Watanabe et al., 1974). According to Doell and colleagues (1981), exceptionally high intakes of TIA from this source are therefore unlikely in the Western diet. Furthermore, the method (a slight modification of the very widely used procedure of Kakade et al. [1969]) used by these researchers to estimate residual TIA of various foods in British diets also was not without limitations. According to Doell and coworkers (1981), the TIA of raw whole soybean (37.7% protein content) was 22.9 mg/g dry sample, i.e., equivalent to 2.29% on a seed weight basis or about 6.1% of protein content. On the basis of the data provided by these researchers (49.6 mg TIA/g protein), TIA still accounted for almost 5% of total soy protein. In contrast, most studies report that the total TIA in soybean

and other food legumes does not exceed 2% to 2.5% of total legume protein (see Liener, 1976; Sgarbieri and Whitaker, 1982; Rackis et al., 1986; and the references cited therein). The literature survey of Rackis and associates (1986) further suggests that most legume species in fact contain less than 50% of the TIA of soybeans (Table 10.4). Particularly low activities are present in most cultivars of fava bean, pea, and lupin and a few of the dry beans. Those with at least 75% of the TIA in soybeans include cowpea, dolichos bean, pigeon pea, and dry beans. Because most household preparations of other legume species include elaborate processing, unlike soybean, which is used primarily as an oilseed rather than a conventional food legume, the likelihood of their residual TIA contribution to human diet appears practically nonexistent. On the basis of these arguments, undue concern about the physiological significance to humans is rather unjustified, although there is little doubt that the proteinase inhibitors from legumes can produce adverse physiological effects in animals. It should also be noted that almost all animal studies that suggest pancreatic hypertrophy as a typical response to a legume-based diet were carried out with either purified inhibitors or raw, unheated legume flours. Both conditions are inconceivable and extremely unlikely in human nutrition. These arguments and rationale become even more complex because the TIA is measured invariably in vitro by its ability to inhibit bovine or porcine pancreatic trypsin under ideal laboratory conditions. In contrast, as mentioned earlier, the human trypsin exists in both a cationic form, which is the major component of human pancreatic juice, and an anionic form, which makes up about 10% to 20% of the total trypsin activity. Although the less active anionic form is inactivated by the soybean trypsin inhibitor in vitro, the predominant cationic species is only weakly inhibited. On the basis of the arguments presented by Deshpande (1992), the earliest cultivated species of food legumes probably contained much higher levels of antinutrients and Darwin’s proclamation that species continually adapt themselves to the evolving environment as a means for their survival, we could even speculate that the distribution of these two forms of trypsin in Homo sapiens is probably a direct result of their adapting legumes as a part of diet in the early history of our civilization. This argument certainly holds true because before learning and devising simple ways of eliminating antinutritive effects of proteinase inhibitors by such methods as soaking, heat treatment, and fermentation, our ancestors must have been consuming TIA at levels high enough to cause fits in any modern-day food scientist and human nutritionist! The two forms of trypsin in humans must therefore have evolved as a direct adaptive mechanism to such excessive intake of TIA in the nomadic diets.


Deshpande (1992) considers the whole issue of nutritional concern over proteinase inhibitor intake from food legumes, scientific curiosity apart, as rather unjustified. Epidemiological surveys of dietary patterns from various regions of the globe prove the safety of food legume consumption in human nutrition. Although early studies suggested that feeding raw legumes to animals results in a considerable loss of protein from the site of its absorption in the small intestine, a direct consequence of the presence of trypsin inhibitors in legumes, they also proved that proper heat treatment of legumes largely alleviates these deleterious effects. Similarly, although these inhibitors constitute only 2% to 2.5% of total protein, they account for about 30% to 40% of the total cystine content of legume proteins (Kakade et al., 1969; Liener, 1976; Deshpande and Deshpande, 1991). Because their heat susceptibility under normal processing conditions has been proved beyond reasonable doubt, food scientists and nutritionists are looking at the possibility of breeding for higher levels of trypsin inhibitors as a means of increasing the Samino acid content and therefore improving the nutritional quality of legumes (see Deshpande, 1992, and the references cited therein). Moreover, proteinase inhibitors and other antinutrients in legumes are possibly involved in plant defense mechanisms against pests and diseases, and eliminating them through breeding approaches may create hitherto unknown physiological and agronomical problems in legume cultivation (Deshpande and Sathe, 1991). Proteinase inhibitors, therefore, seem to cause few, if any, deleterious effects in human nutrition.

10.3 AMYLASE INHIBITORS Naturally occurring inhibitors of pancreatic amylase were first discovered in aqueous extracts of wheat, rye, and kidney beans (Kneen and Sandstedt, 1943; Bowman, 1945). Jaffe and associates (1973) reported the presence of αamylase inhibitory activity in 79 of 95 legume cultivars tested; the most activity was found in a kidney bean (Phaseolus vulgaris) cultivar. Deshpande and colleagues (1982) also reported substantial α-amylase inhibitor activity in several cultivars of dry beans. The physiological role of α-amylase inhibitors in plants is not well understood. They are not active against the endogenous α- and β-amylases of legumes or those in malt, barley, or microbial amylases (Jaffe et al., 1973; Powers and Whitaker, 1977a). The bean inhibitors, however, inhibit insect larva α-amylase and, therefore, may have a physiological role in protecting the seeds against insect attact (Sgarbieri and Whitaker, 1982; Deshpande and Sathe, 1991).

Amylase inhibitors have been purified to homogeneity from kidney beans (Marshall and Lauda, 1975; Powers and Whitaker, 1977a). They constitute about 5% to 6% of the total water-soluble protein in kidney beans. Their molecular weights range from 45,000 to 49,000 Da, and all appear to be glycoproteins containing 8% to 10% carbohydrates. On SDS-PAGE, the protein gives four subunits of three size classes of molecular weights 15,000 to 17,000, 12,000 to 15,000, and 11,000 to 12,000 Da. The inhibitors have no Pro and two Cys residues and are relatively rich in Trp, Tyr, Val, and Gly. The kidney bean amylase inhibitors form a 1:1 complex with pancreatic α-amylase (Marshall and Lauda, 1975). However, the specific groups involved in the complex formation and the mechanism of inhibition are not known. The carbohydrate portion of the inhibitor appears to play a crucial role in this regard, since its periodate oxidation results in a complete loss of inhibitor activity (Powers and Whitaker, 1977b). Although the complex has no activity, it can still bind to maltose. This observation led these researchers to speculate that a binding site of α-amylase is still available in the inhibitor-enzyme complex, although by itself it is catalytically inactive. On a nutritional level, amylase inhibitors appear to affect the rate of mammalian starch digestion (Pace et al., 1978). However, this effect has been controversial, since Kneen and Sandstedt (1946) concluded that amylase inhibitors from wheat are inactivated by pepsin and, thus, are not of much nutritional significance. Some evidence has indicated that large amounts of the inhibitor may overcome gastric digestion in laboratory animals and humans (Puls and Keup, 1973). This has not been confirmed in several laboratory animal and most clinical studies (Savaiano et al., 1977; Carlson et al., 1983). Since amylase inhibitors are quite heat-stable, they have been detected in significant amounts in baked wheat flour products and some wheat-based breakfast cereals (Marshall, 1975). However, relatively few studies have been carried out on the physiological effects of these residual inhibitors. Evidence is conflicting as to whether amylase inhibitors reduce growth in animals. In one study in rats, wheat-derived amylase inhibitor reduced growth rate, whereas heat-inactivated inhibitor showed no adverse effect (Saunders, 1975). On the contrary, Savaiano and coworkers (1977) found kidney bean inhibitor to be quite ineffective in this regard. The source of the inhibitor may explain the discrepancy between the findings of these two studies. A decreased rate of starch hydrolysis due to the inhibitors in the small intestine would logically lead to a slower absorption of glucose and possibly cause certain metabolic changes. In rats and dogs, a wheat-derived amy-


lase inhibitor added to a starch diet attenuated both a rise in blood glucose level and a fall in nonesterified fatty acids level that normally occur in such diets (Puls and Keup, 1973). In humans, a similar effect of the inhibitor on blood glucose levels was observed. Long-term feeding of this type of inhibitor led to a decreased rate of incorporation of 14 C starch into lipids of epididymal adipose tissue and of aortic tissue. Since proteinase inhibitors increase the pancreatic trypsin and chymotrypsin levels, one might expect amylase inhibitors to produce an analogous response. However, regulation of pancreatic amylase levels appears to be functionally different from that of the proteinases. The former is related to the quantity of digestion products absorbed (Lavau et al., 1974). Amylase inhibitors were also critically evaluated as to their effectiveness in the treatment of metabolic diseases, such as diabetes and adiposity (Fukuhara et al., 1982). On the basis of in vitro and early in vivo evidence that ingested kidney bean amylase inhibitors could block the digestion of starch in meals, starch blocker tablets containing these inhibitors were sold for weight control. However, Carlson and associates (1983) found several reasons for the apparent commercial lack of effect of starch blockers. These included the inactivation of the inhibitor by gastric acid and pepsin or by pancreatic proteinases, intraluminal conditions unfavorable to maximal inhibitions, and insufficient preincubation time for the inhibitor. However, the potential of new starch blockers for therapeutic purposes, such as in treating diabetes and obesity, merits long-term research.

10.4 LIPASE INHIBITORS In contrast to the large number of known proteinase and, to some extent, amylase inhibitors of plant origin, virtually no lipase inhibitors have been identified. Both Mori and associates (1973) and Satouchi and colleagues (1974) reported a protein from soybean cotyledons that decreased the lipolytic activity of a pancreatic lipase. Since all lipases primarily act at the oil-water interface of an emulsified oil droplet, the substrate concentration is dependent upon the total interfacial area. Inhibitors of pancreatic lipase may act as true inhibitors by forming the enzyme-inhibitor complex, or they may reduce the enzyme activity by adhering to the oil-water interface, thereby effectively reducing the substrate concentration. Subsequent studies on the soybean cotyledon lipase inhibitor revealed its mode of action to be adherence to the interface (Satouchi and Matsushita, 1976) and not enzyme inhibition in the

classic sense. Whether this protein would have any physiological significance remains to be explored.

10.5 LECTINS (PHYTOHEMAGGLUTININS) Lectins have been identified in many plant families, from slime molds, fungi, and lichens to flowering plants (Jaffe, 1980; Sharon and Lis, 1989; Liener, 1997). They are also found in animals, such as sponges, crustaceans, and mollusks; and in fish blood serum, amphibian eggs, and even mammalian tissue. Among several species of over 140 plant families, Toms and Western (1971) reported 79 hemagglutinin-positive species. According to Liener (1976), in over 800 different plant species in which lectin activity has been detected, more than 600 belong to the Leguminosae family. In many edible legumes, lectins thus are of widespread occurrence, and they constitute between 2% and 10% or more of the total seed protein. The presence of lectin was recognized as early as 1888, when Stillmark observed that the extreme toxicity of the castor bean could be attributed to a protein fraction, which was capable of agglutinating red blood cells (Stillmark, 1888). He coined the name ricin for this substance because it was derived from Ricinus communis. Lectins are a group of natural products that show some apparently unrelated characteristics. Their only common feature is that they are all proteins or glycoproteins. Their specificity and effects are measured in agglutination tests with treated or untreated erythrocytes, from which the term lectin (legere, “to elect, to choose”) is derived (Liener, 1997). In addition to agglutinating red blood cells, lectins exhibit a number of other interesting and unusual

biological and chemical properties, including interaction with specific blood groups, mitogenesis, agglutination of tumor cells, and toxicity to animals (Sharon and Lis, 1989). All of the effects manifest their ability to bind to specific kinds of sugars on the surface of cells. Their reaction mechanism is comparable to that of human antibodies, but lectins are not induced by or the result of an immune response. Several aspects of the chemical and biological properties of various plant lectins have been comprehensively reviewed (Liener, 1976, 1997; Pusztai, 1989; Sharon and Lis, 1989). Examples of the properties of some of these lectins are listed in Table 10.9. On the basis of their sugar-binding specificity, lectins are classified into several groups. Lectins binding D-mannose and D-glucose are found in the seeds of Pisum, Vicia, Lens, and Canavalia spp. and are mitogenic to lymphocyte (Jaffe, 1980). N-acetyl- D -galactosaminebinding lectins isolated from Glycine spp. and Phaseolus lunatus are specific for blood group A, and in the case of lima beans, they are also mitogenic. Arachis lectin is a Dgalactose-binding protein that agglutinates type B erytrocytes. Phaseolus vulgaris produces lectins with complex carbohydrate-binding sites (Pusztai, 1989). Thus, there are obviously no recognizable patterns between the chemical and biological properties of the lectins and their taxonomic distribution in the Leguminosae family. Lectins with the same specificity are therefore found in different genera, and within one genus, both specific and nonspecific lectins are found. There is a great variability in the biological effects of lectins, especially in the genus Phaseolus (Brown et al., 1982a, 1982b; Felsted et al., 1981a, 1981b). These biological effects are associated with different polypeptides. This

Table 10.9 Biochemical Properties of Selected Lectins

Botanical name

Common name

Molecular weight

Number of subunits

Arachis hypogaea Canavalia ensiformis Dolichos biflorus Glycine max Lathyrus odoratus Lathyrus sativus Lens esculenta Phaseolus lunatus Phaseolus vulgaris Pisum sativum Psophocarpus tetragonolobus Vicia faba

Peanut Jack bean Horse gram Soybean Sweet pea Chickling vetch Lentil Lima bean Kidney bean Pea Winged bean Fava bean

110,000 105,000 110,000 120,000 52,000 49,000 46,000 60,000 126,000 49,000 58,000 52,500

4 4 4 4 4 4 4 2 4 4 2 4

a

GalNAc, N-acetylgalactosamine; Man, mannose. Source: Compiled from Deshpande and Deshpande (1991) and Liener (1997).


Sugar specificitya GalNAc Man GalNAc GalNAc Man Man Man GalNAc GalNAc Man GalNAc Man

may explain the observed variability in agglutinating and mitogenic activities, since the polypeptide composition may vary in protein preparations from the same source. The ability to agglutinate erythrocytes necessitates polyvalent binding sites. Most lectins appear to have molecular weights in the range of 100,000 to 150,000 Da and are composed of tetramers. Some, such as lentil and lima bean lectins, appear to be dimers. With few exceptions (e.g., soybeans), each subunit has a sugar-binding site (Liener, 1997). The hybrid tetramer consists of erythrocyte-reactive subunits (E) and lymphocyte-reactive subunits (L) (Felsted et al., 1981b). The five possible isolectins would have the tetrameric structure: L4, L3E1, L2E2, L1E3, and E4, which explain the differences in the observed agglutinating and mitogenic activities of bean lectins. This property is lost if the lectins are dissociated into subunits. Most, if not all, lectins also contain up to 4% to 10% carbohydrates. Data regarding the biological functions of lectins still appear to be contradictory. The ability of lectins to bind to characteristic carbohydrate structures on the cell surface and the observed variability in lectin production in different genotypes of legumes have initiated a number of systematic investigations of the functions of lectins in host plants. Lectins appear to be important determinants of host-range specificity in Rhizobium sp.–legume symbiosis (Sharon and Lis, 1989). Thus, the lectin of Glycine sp. binds only to symbiotic strains of Rhizobium sp., although it is probably not essential for initiating the symbiotic relationship. Another biological function ascribed to the lectins is that of an insecticide. This function is interpreted in terms of an adaptive significance of the lectins in Phaseolus vulgaris for protecting seeds from attack by insect seed predators. One interesting aspect was discussed in connection with the characterization of a lectin isolated from the seeds of Phaseolus aureus, which possesses a strong enzymatic activity (Hankins and Shannon, 1978). By comparing data concerning the synthesis, distribution, and function within plants, it was postulated that legume lectins might, in general, be plant enzymes. However, the lack of detailed information on different aspects of the biological functions of lectins raises more questions that still need to be answered. Lectins were among the first toxic factors implicated in the toxicity of raw legumes to laboratory animals. Earlier studies in this regard were done in the laboratories of Liener. On the basis of observations that the antitryptic factor produced growth depression even when added to a ration containing a protein hydrolysate, Liener and associates (1949) suggested the presence in soybeans of a “substance other than the antitryptic factor which adversely affects growth.” Further investigations resulted in the isolation from raw soybean meal of a very potent hemag-


glutinating protein fraction (Liener, 1951; Liener and Pallansch, 1952). When this fraction was added to a ration containing autoclaved soybeans, the rats gained weight at a rate equal to 75% that of the rats fed the basal ration (Wada et al., 1958). The primary effect of the hemagglutinin, as far as growth was concerned, appeared to be a depression of appetite. Liener (1958) also reported that the hemagglutinating activity was readily inactivated by pepsin. Since these earlier studies, several researchers have noted a marked reduction in protein digestibility and a growth inhibitory effect derived by depressing the appetite when purified lectins are added to the diet. As far as the mode of action is concerned, Jaffe (1980) postulated that the toxic effects of lectins when ingested orally may be due to their ability to bind to specific receptor sites on the surface of intestinal epithelial cells, thus causing a nonspecific interference with the absorption of nutrients. The fact that lectins are so widely distributed in food items commonly consumed by humans raises the important question as to whether they pose any significant risk to human health. Fortunately, most lectins are easily destroyed by the traditional methods of household cooking. As an example, the effectiveness of processing on the lectin content of food items containing soybeans as an ingredient is shown in Table 10.10. It is doubtful whether such low levels of lectin activity pose a risk to human health. Thus, there would be little cause for concern in human nutrition. Nevertheless, under special conditions, complete detoxification may not always be achieved, especially if ground seeds are used or industrial processes for quickcooking products are applied. Since lectins are resistant to inactivation by dry heat (Jaffe, 1980), practices such as the use of raw legume flours in baked goods should be viewed with caution. Of greater concern are the toxic effects that are associated with the inadvertent consumption of beans and

Table 10.10 Lectin Content of Processed Foods Containing Soybeans as an Ingredient Product Raw soybeans Unprocessed soy flour Defatted soy flour Textured meat analog Breakfast cereal Soy milk Cookie

Raw soy, % 100.0 47.0 4.3 0.4 5.2 0.5 0.7

Source: Compiled from Liener (1997).

dishes derived from them that have been improperly cooked or processed. Lectins appear to have been the causative agent in several cases of human intoxification. These symptoms are primarily manifested as gastrointestinal discomforts. An outbreak of what appeared to be food poisoning occurred in England in 1976. A party of schoolboys on holiday ate kidney beans that had been soaked in water but had not been cooked. All nine of the boys who ate the beans became acutely nauseated within 60–90 minutes and began to vomit, then to have diarrhea (Liener, 1977). Eating as few as four to five beans was sufficient to produce these reactions. Two of the boys were admitted to the hospital and needed intravenous infusion. However, recovery was rapid in all cases. Illness after the consumption of raw beans as part of a salad or in dishes such as a stew, casserole, or chili con carne cooked in a slow cooker is not uncommon. The times and temperatures involved in the slow cooking of kidney beans under household conditions may not be sufficient to destroy all of the lectin activity. Warning labels are often found on labels of dried kidney beans sold in the retail food markets in England and several other European countries, recommending that the beans be boiled for at least 10 minutes before consumption. Because of their unique capacity to bind in a specific fashion to sugars and other glycoconjugates, the lectins have broad applications in research and biomedical laboratories (Liener, 1997). Either in solution or in an immobilized form, lectins have proved extremely useful for the detection and identification of many diverse glycoconjugates. The identification of blood group substances, and membrane receptors and the detection of malignant cells are examples of clinical applications of lectins. Lectins could also be used to prevent graft rejection in bone marrow transplantation (Sharon and Lis, 1989). This potential derives from the property that the soybean lectin can be used to remove the mature T cells responsible for graft rejection.

10.6 PHYTATE Cereals and legumes contain significant amounts of phosphorus in the form of phytic acid (myo-inositol hexaphosphate). The terms phytic acid, phytate, and phytin refer to free acid, salt, and Ca2+/Mg2+ salt, respectively; however, these terms are often used interchangeably. The revised nomenclature for phytic acid in plant seeds is myo-inositol-1,2,3,5/4,6-hexakis (dihydrogen phosphate) (IUPACIUB, 1968). Phytic acid occurs primarily as a salt of mono- and divalent cations in discrete regions of cereal grains and legumes. It rapidly accumulates in seeds during the ripening


period in the electron-dense aleurone particles or globoids (Reddy et al., 1982). Phytate accounts for up to 85% of the total phosphorus in many cereals and legumes. The phytate phosphorus and phytate contents of selected foodstuffs are summarized in Table 10.11.

Table 10.11

Food

Phytate Content of Selected Foodstuffs

mg %

Cereals Wheat 170–280 Rye 247 Maize 146–353 Rice 157–240 Barley 70–300 Oats 208–355 Sorghum 206–280 Buckwheat 322 Millet 83 Wheat bran 1170–1439 Legumes and vegetables Dry beans 269 Lima beans 152 Soybean 402 Lentil 295 Pea 117 Vetch 500 Chickpea 140–354 Pea 82 Potato 14 Green bean 52 Green pea 12 Carrot 0–4 Nuts and seeds Walnut 120 Hazelnut 104 Almond 189 Peanut 205 Cocoa bean 169 Pistachio 176 Rapeseed 795 Cottonseed 366 Spices and flavoring agents Caraway 297 Coriander 320 Cumin 153 Mustard 392 Nutmeg 162 Black pepper 115 Pepper 56 Paprika 71

Phytate phosphorus, % of total phosphorus 47–86 73 52–97 68 32–80 50–88 77–88 70 57 89–97 62 77 65 90 37 95 49–95 31 35 43 12 0–16 24 45 43 57 25 75 89 41 96 77 33 86 61 58 15 15

Source: Compiled from Concon (1988) and Deshpande and Sathe (1991).

The presence of phytate in processed foods has received considerable attention in recent years. Phytate is generally considered to be fairly heat-stable. Among the processing methods, germination and fermentation appear to be quite effective in decreasing the phytate concentrations, whereas soaking and cooking can remove more than 50% to 80% of the endogenous phytate in legumes and cereals (Deshpande and Damodaran, 1990; Deshpande et al., 1984a). The concern about phytate-mineral interactions is caused by the ability of phytate to form insoluble complexes with minerals at physiological pH values. Phytic acid has six reactive phosphates and meets the criterion of a chelating agent. In fact, a cation can complex not only within one phosphate or between two phosphate groups of the phytic acid, but also between two phytic acid molecules (Erdman, 1979). Thus, phytate is largely blamed for complexing dietary essential minerals in legumes and cereals and rendering them poorly available to monogastric animals. Zinc and copper appear to bind phytate in the physiological pH range more tightly than other minerals (Maddaiah et al., 1964). The possible interactions of phytate with minerals and with protein and starch are shown in Figure 10.1. The mechanism by which phytate affects mineral bioavailability is not clearly understood. Several studies, however, suggest the formation of insoluble phytate-mineral complexes in the intestinal tract, which prevent mineral absorption (Graf and Eaton, 1984; Wise, 1983;

HO O P

Ca2+

O

O- -O

O

Starch

O

-

-O

-O

O-

C

CH2-Protein

O

CH2 NH3

O

+Ca+

O P OH O

Protein

+

P O

O P OH O P O O

O-O

P O

HO CH2OH O O

O

n

Starch

Figure 10.1 Possible interactions of phytic acid with minerals, proteins, and starch.


Erdman, 1979). The formation of these complexes is pHdependent. However, in most cases, the bioavailability of minerals in laboratory animals has been studied by using free ionic salts. The results obtained under such conditions may not represent the true bioavailability of minerals from similar sources to humans. The reduced bioavailability of minerals from phytate-rich sources depends on several factors, which include the nutritional status of animals/humans, the concentration of minerals and phytate in foodstuffs, the ability of endogenous carriers in the intestinal mucosa to absorb essential minerals bound to phytate and other dietary substance, the digestion or hydrolysis of phytate by phytase and/or phosphatase enzymes, the processing of products or methods of processing that include unit food processing operations (pH adjustment, level of refinement, and addition or removal of inhibitors/enhancers), and the digestibility of the foodstuffs (Reddy et al., 1982; Deshpande et al., 1984a; Deshpande and Deshpande, 1991). Phytate is not the only dietary component that influences mineral bioavailability. Other food components, such as dietary fiber, polysaccharides, oxalates, and polyphenols, are also known to influence the bioavailability of minerals adversely. In addition to complexing metal ions, phytate interacts nonspecifically with various proteins, enzymes, and carbohydrates (Figure 10.1). Phytate inhibits several important enzymes, such as trypsin, pepsin, α-amylase, and β-glucosidase. Sharma and colleagues (1978) studied the phytate inhibition of α-amylase derived from several plant sources. The inhibition of wheat α-amylase was found to be noncompetitive, with an apparent Ki of 1 mM. Later, Deshpande and Cheryan (1984) observed a 16% to 95% decrease in activity of a porcine pancreatic α-amylase at 0.5- to 6-mM phytate concentration. Conflicting mechanisms have been proposed for phytate inhibition of amylases. Cawley and Mitchell (1968) suggested phytate chelates Ca2+ ions, which are activators of amylases in sprouted wheat meal. On the contrary, Sharma and coworkers (1978) found that the addition of Ca2+ did not reverse the inhibition and that the inhibition was related to the direct interactions of phytate with the enzyme allosteric site. Subsequently, Deshpande and Cheryan (1984) suggested that the general complex-forming ability of phytate with enzyme proteins was the major reason for the inhibition of amylases. That phytate should inhibit proteolytic enzymes such as pepsin (Knuckles et al., 1985) is not surprising, since the acidic pH at which it is active can promote strong electrostatic linkages between phytate and the positively charged groups of protein. Indeed, the same can be said of enzymes whose pH optima are on the acid side of the

scale. In contrast, the more negative charges on proteins at neutral to alkaline pH may prevent formation of phytateprotein binary complexes unless an external povalent cation (such as Ca2+ or Mg2+) is present. Thus, phytate inhibition of proteolytic enzymes, such as trypsin and chymotrypsin, is insignificant compared to that of pepsin. In a study on the inhibition of trypsin activity in vitro by phytate, Singh and Krikorian (1982) reported 20% enzyme inhibition at 90 mM phytate in the assay system. On the basis of the data provided by these authors, this turns out to be an almost 3000:1 phytate-enzyme ratio. Deshpande and Damodaran (1989c) have argued that not only are such ratios of free phytate difficult to encounter under in vivo conditions, but also because of their high ionic strength, they may effectively salt out the enzyme from the assay system. Deshpande and Damodaran (1989c), using phytate-enzyme ratios that are more likely to be encountered under normal in vivo conditions, in fact observed a 5% to 7% increase in the activity of both trypsin and chymotrypsin. Only one other report in the literature suggests a positive influence of phytate on enzyme activity; Altschuler and Schwartz (1984) reported an enhancement of both mutant and wild-type enzyme activity of alcohol dehydrogenase from corn in the presence of phytate. The interaction of phytate with enzymes may be important in human nutrition, since it may result in decreased protein and starch digestibility of foods rich in phytate. Thompson and coworkers (Yoon et al., 1983; Thompson and Yoon, 1984) studied the effects of phytate on the in vitro starch digestibility by human saliva enzymes, as well as the blood glucose response (glycemic index) in healthy volunteers. A significant negative correlation between phytic acid (both concentration and intake) and the glycemic index was observed. Foods that were rich in phytate were digested at a slower rate and produced lower blood glucose responses than foods devoid of phytate. Button and associates (1985) found that the removal of phytate produces an increase in the rate of starch digestion both in vitro and in vivo, whereas the readdition of phytate yields an opposite effect. Thompson (1988) even suggested that there may be a therapeutic role for phytate in the management of diabetes and obesity and that complete elimination of phytate during processing may actually be undesirable. However, the levels at which phytate combines maximal health benefits with negligible adverse effects have not yet been determined. Although this seems to be a research area of considerable potential, the strategy of not eliminating phytate appears to be questionable, particularly in the developing countries, where the consumption of phytaterich legumes is high and at the same time malnutrition is prevalent.


As compared to carbohydrate metabolism, phytate does not seem to influence dietary protein utilization to any significant extent. Several studies have clearly shown that although the digestibility of protein and the subsequent rate of release and absorption of amino acids are only marginally higher in low-phytate diets, from phytate complexed proteins they are not significantly different (Thompson and Serraino, 1986; Thompson, 1988; Reddy et al., 1988). Then there is also the question of how much “free” phytate would be available to inhibit the digestive enzymes. In addition, most in vitro enzyme inhibition studies use sodium phytate, whose solubility behavior is completely different from that observed for phytate from plant sources. Phytic acid is a highly unstable molecule and in nature is always present as a salt of Ca2+, Mg2+, or K+. When one considers its strong affinity to various cations and the type of interactions involved in its association with dietary protein as well as factors such as food processing, thermal degradation of inositol esters, and pH, little free phytate would be expected to be available to interact with enzymes in the digestive system and have any significant influence. In addition, it has been clearly shown that in the presence of minerals such as Ca2+ or Mg2+, the actual in vitro inhibition of enzymes would be much lower (Deshpande and Cheryan, 1984). Similarly, hosts of different enzymes are involved in the digestion process. Proteins in which some lysyl or arginyl side chains are complexed with phytate may not be effectively hydrolyzed by trypsin; under these conditions, enzymes such as chymotrypsin would prove more effective, since the latter shows specificity for large hydrophobic side chains. Until several questions regarding phytate-protein interactions under a wide variety of conditions and their influence on protein digestibility under simulated in vivo conditions are addressed and answers that are more definitive found, the adverse effects of phytate in human nutrition, given its nature and chemical properties, should be strictly addressed from the mineral bioavailability point of view. Rickard and Thompson (1997a) have suggested a protective role for phytic acid in the prevention of cancer of the colon, mammary gland, and other organs or tissues at various stages of the carcinogenic process. The ability of phytic acid to bind starch, proteins, and minerals has been implicated as part of the mechanism whereby phytic acid exerts its anticancer effects, although there is evidence that the hydrolysis products of phytic acid also play an important role. One of the breakdown products of phytic acid is inositol triphosphate (InsP3), which acts in cellular signal transduction and in the enhancement of natural killer (NK) cell activity. Phytic acid does not appear to act through a cytotoxic mechanism but through promotion

of the differentiation of the malignant cells to a more normal phenotype. In light of its healthful properties, Rickard and Thompson (1997a) suggested that the term antinutrient is an outdated label for food constituents like phytic acid.

10.7 TANNINS (POLYPHENOLS) Phenolic compounds (i.e., phenolic acids and their derivatives) are widely distributed in the plant kingdom. Although the pharmacological and therapeutic properties of many bioflavonoids (a class of phenolic compounds) are well documented in the literature, there is no evidence that they have a nutritional role in our diet. Plants provide nearly all the phenols to higher animals, since the latter cannot synthesize compounds with benzenoid rings from their aliphatic precursors. Phenolics are thus strictly exogenous food components of exclusively vegetable origin. The common dietary low-molecular-weight phenolics, owing to the evolution of efficient detoxification, are not considered toxicants under normal amounts and conditions. The condensed tannins of flavonoid origin (also referred to as polyphenols) are one possible exception. They are widespread in fruits and vegetables and in certain grains. The pigmented varieties of certain cereals and legumes contain 2% to 4% condensed tannins, although amounts as high as 7% to 8% have been reported for red high-tannin sorghum varieties (Mabbayad and Tipton, 1975). Humans also consume a number of other foods containing considerable amounts of condensed tannins, especially in beverages, such as cider, tea, cocoa, and red wine. Strong versions of these beverages may have as much as 1 g tannin per liter. The tannin content of several cereals and legumes is summarized in Table 10.12. The intake of dimeric flavans may be up to 400 mg/day in human diets (Kuhnau, 1976). The total tannin intake would be somewhat higher. The per capita consumption of red wines in some countries would guarantee an average intake of nearly that amount from that source alone (Singleton, 1981). Rao and Prabhavati (1982) reported a range of 1.5 to 2.5 g for the daily intake of dietary tannins in different regions of India. Tannins comprise a heterogeneous group of plant polyphenols all of which are able to combine with skin proteins in such a way as to render them resistant to putrefaction, or, in other words, to “tan” them into leather. More specifically, they are high-molecular-weight compounds (500 to 5000 Da) containing sufficient phenolic hydroxyl groups to permit the formation of stable cross-links with proteins. Although the presence of ortho dihydroxy phenols seems to be essential (Hathaway and Seakins, 1958;


Table 10.12 Tannins of Cereals, Millets, and Legumes Source Cereals and millets Sorghums Wheat Corn Finger millet Legumes Pigeonpeas Chickpeas Soybeans Green gram Lima beans Black gram Dry beans Peas Winged beans Fava beans Cowpeas Adzuki beans

Tannins, % 0.04–7.87 0.12–0.41 0.13–0.14 0.36–1.17 0.03–1.00 0.03–0.22 0.045 0.03–0.47 0.77 0.31–0.86 0.00–2.00 0.43–0.47 0.403 0.75–1.92 0.19–2.12 0.29–0.37

Source: Compiled from Deshpande et al. (1986) and Deshpande and Damodaran (1990).

Bauer-Staob and Niebes, 1976) presumably to form hydrogen bonds with groups on the proteins, hydrophobic binding may be an important contribution to the stability of the complex. Tannins usually give rise to a dry, puckery, astringent sensation in the mouth. Because of their proteinbinding properties, tannins are of considerable importance in food processing, fruit ripening, and manufacturing of tea, cocoa, and wine. On the basis of structural types, tannins have been classified by Freudenberg (1920) into two groups: the hydrolyzable tannins and the condensed tannins. The condensed tannins are more widely distributed in higher plants. Most hydrolyzable tannins contain a central core of glucose or other polyhydric alcohol esterified with gallic acid (gallotannins) or hexahydroxydiphenic acid (ellagitannins). The latter is isolated as its stable dilactone, ellagic acid. These types of tannins are readily hydrolyzed by acids, bases, or certain enzymes. The condensed tannins, also referred to as procyanidins and, formerly, leucoanthocyanidins because many form cyanidin on acid hydrolysis, are mostly flavolans or polymers of flavan-3-ols (catechins) and/or flavan-3,4-diols (leucoanthocyanidins). Both are readily converted by dehydrogenating enzymes or even by very dilute mineral acids into flavonoid tannins at room temperature (Weinges, 1968). The condensed tannins are not readily degraded by

acid treatment but polymerize to form amorphous phlobaphenes or “tannin-reds.” Most physiological investigations of dietary tannins have been made with nonruminants. When fed at levels that commonly occur in cereals and legumes (up to 1% to 2%), tannins have depressed the growth rate, resulted in a poor feed efficiency ratio, and increased the amount of food required per unit weight gain (Price and Butler, 1980; Deshpande et al., 1984b). Other deleterious effects of tannins include damage to the mucosal lining of the gastrointestinal (GI) tract, alteration in the excretion of certain cations, and increased excretion of proteins and essential amino acids. High dietary levels (about 5%) can cause death (Singleton and Kratzer, 1969). The deleterious effects of tannins in the diet seem to be related to their interactions with dietary proteins. Tannin-protein complexes are believed to be responsible for growth depression, low protein digestibility, and increased fecal nitrogen level. Casein, bovine serum albumin, G1 protein from beans, and carob pod proteins resist proteolytic digestion when complexed with tannins (Feeney, 1969; Deshpande, 1985; Tamir and Alumot, 1969). Such complexes may not be dissociated at physiological pH and may pass out in the feces. The nitrogen content of the feces generally rises in proportion to the amount of tannin fed. Tannins also inhibit digestive enzymes, such as trypsin and amylases (Davis and Harbers, 1974; Deshpande and Salunkhe, 1982; Tamir and Alumot, 1969). Tannins have been implicated in carcinogenesis. Epidemiological studies conducted on a global basis suggest a possible correlation between unusual consumption of plant materials rich in condensed tannins (particularly high-tannin sorghums and the dark beers prepared from them, tea, red wines, and areca nuts in certain parts of the world) and the unusual frequency of cancer of the esophagus and mouth (Morton, 1970, 1972). Tannins and tannic acid have also been listed as tentative carcinogens of category I under the general carcinogenic policy of OSHA (OSHA, 1978). Several methods to overcome the antinutritional effects of tannins in the diet have been attempted. The basic principles involved include the physical removal of tannins by extraction or milling, addition of agents that complex with dietary tannins, use of agents that aid in the metabolic detoxification of tannins, and plant breeding approaches. Since tannins are primarily located in the seed coats, dehulling appears to be the simplest and least expensive approach to removing dietary tannins from food grains; however, the beneficial effects may be partially offset by some nutrient losses. Most food processing methods, such as soaking, cooking, and germination, result in an apparent loss of measurable tannins. A long-term approach, and the


most satisfactory one, would be to breed for varieties with fewer or no tannins. In recent years, several media reports comparing the dietary habits of French and U.S. people have ascribed certain beneficial effects such as lowering of the frequency of blood-related disorders to regular consumption of red wines in the French diet. Although moderate alcohol intake seems to have beneficial effects in human nutrition, the consumption of red wines is also accompanied by increased intakes of phenolics that should not be ignored in the French diet. The anticarcinogenic activities of polyphenols in foods and herbal medicines were reviewed by Miyamoto and colleagues (1997). Among the major tannin activities found are antioxidant and radical scavenging activities. These are the basic activities underlying the effects of tannin-rich medicinal plants that are effective in preventing and treating many diseases such as arteriosclerosis, heart dysfunction, and liver injury, as well as inhibiting lipid peroxidation. The inhibition of hepatotoxins and mutagens and the antitumor-promoter action of polyhphenols are also correlated with their antioxidant activity. In this regard, oligomeric ellagitannins, which consist of monomer units such as potentillin, tellimagrandin I and II, and related structures, seem to have potent anticancer activities.

10.8 CYANOGENIC GLYCOSIDES Cyanogenic glycosides are important natural toxicants in both animal and human nutrition and are widespread in the plant kingdom. Chronic toxicological effects occur in humans who consume cassava in tropical countries; cyanide production potential is of concern in several other food crops. Livestock poisonings are also associated with the consumption of forage sorghums. Cyanogenic glycosides have been identified in over 2000 species of higher plants belonging to 110 different plant families, including ferns, gymnosperms, and angiosperms (Poulton, 1983; Deshpande and Sathe, 1991). Among these families, the most notable for their cyanogenic ability are Rosaceae (150 species), Leguminosae (125), Graminae (100), Araceae (50), Compositae (50), Euphorbiaceae (50), and Passifloraceae (30). The distribution of some well-characterized cyanogens is summarized in Table 10.13. Cyanogenic glycosides are compounds that on treatment with acid or appropriate hydrolytic enzymes produce hydrocyanic acid (HCN). These compounds in higher plants are of two types: Cyanogenic glycosides usually contain glucose as their sugar component, although other mono- and disaccharides may be found. Cyanogenic lipids are another group of cyanide precursors, which contain,

Table 10.13 Glycoside Amygdalin Dhurrin Linamarin Lotaustralin Prunasin Cicianin

Distribution and Hydrolytic Products of Some Cyanogenic Glycosides in Food Plants Plants

Hydrolytic products

Almond, cherry, peach, plums, apples Sorghums Linseed, clovers, cassava, lima beans Linseed, cloves, cassava, lima beans Cherry, almond Vetches

HCN, gentobiose, and benzaldehyde HCN, glucose, and hydroxybenzaldehyde HCN, glucose, and acetone HCN, glucose, and 2-butanone HCN, glucose, and benzaldehyde HCN, vicianose, and benzaldehyde

Source: Compiled from Tewe and Iyayi (1989) and Poulton (1983).

instead of sugar, long-chain fatty acid moieties. Both are derivatives of α-hydroxynitriles (cyanohydrins), and both liberate a carbonyl compound and HCN when, respectively, the sugar or the fatty acid moieties are removed. The chemical structures of some well-characterized cyanogenic glycosides are shown in Figure 10.2. The cyanogenic glycosides are relatively stable chemical compounds at neutral pH. They can be hydrolyzed to their component parts (an aldehyde or ketone, sugar, and HCN) by acid at elevated temperatures. The production of HCN from cyanogenic glycosides is an enzymatic process, commonly known as cyanogenesis. One of the best-studied examples is that of linamarin, which is found in lima beans and cassava. In these plants, the presence of an endogenous enzyme linamarinase or linase causes its hydrolysis, with the production of HCN. The reaction proceeds via two steps (Figure 10.3). In the first, the linamarin is hydrolyzed by linamarinase to produce β-Dglucopyranose and 2-hydroxyisobutyronitrile or acetone cyanohydrin. The latter dissociates, catalyzed by a hydroxynitrile lyase, to produce acetone and HCN. Another well-studied example of cyanogenesis is the hydrolysis of amygdalin and prunasin (Figure 10.4). The process involves the stepwise removal of glucose by the action of two separate specific β-glucosidases and the subsequent action of a hydroxynitrile lyase, which catalyzes the dissociation of the α-hydroxynitrile. Since the cyanogenic glycosides accumulate in significant quantities in plant tissues, which also contain these enzymes, it is quite likely that the substrates are sequestered separately from their catabolic enzymes in one part of the plant cell or even in separate tissues (Conn, 1979). This indeed is true for sorghum leaves, where dhurrin is located exclusively in the vacuoles of epidermal cells and the degradative enzymes are found only in mesophyll tissue (Kojima et al., 1979). Cyanogenesis most frequently occurs when a cyanogenic plant tissue is crushed or otherwise disrupted (Conn, 1979). This may occur during the processing (grinding, drying, pounding) of the plant tissue during food prepara-


tion and obviously takes place when the plant tissue is ingested directly and chewed by an animal. To the extent that the catabolic enzymes could be removed or inhibited in their action, or to the degree that the substrates could be removed or destroyed without the release of HCN, the cyanogenic phenomenon could be reduced in intensity or even prevented. The lethality of cyanide is due to its ability to inhibit respiration. Cyanide is a potent inhibitor of the cytochrome oxidase of the respiratory chain. Cyanide also inhibits several other metalloenzymes, including nitrate reductase, nitrogenase, and xanthin oxidase (all molybdoenzymes); alkaline phosphatase and carbonic anhydrase (zinc enzymes); plastocyanin and ascrobate oxidase (copper enzymes); and some selenoenzymes. Enzymes without metals, such as glutamate decarboxylase and α-amino butyric acid transaminase, can also be inhibited by cyanide (Deshpande and Sathe, 1991). The ability of cyanogenic glycosides to release cyanide implicates them in a number of diseases encountered in populations who depend primarily on food sources rich in these compounds. The consumption of cassava has been reported to cause neurological and endocrinological diseases in many tropical countries (Tewe and Iyayi, 1989). Ingested cyanide is rapidly absorbed from the upper GI tract. It also passes readily through the skin, and HCN gas is rapidly absorbed from the lungs. The amount of HCN produced by different plant species varies considerably (Table 10.14). Proper processing of foods, which usually involves soaking, cooking, and fermentation, is known to reduce the incidence of cyanide poisoning. The minimal lethal dose of HCN for humans has been estimated at between 0.5 and 3.5 mg/kg body weight. This is equivalent to 30 to 210 mg for a 60-kg adult. HCN is readily absorbed from the gastrointestinal tract and produces recognizable symptoms at both lethal and sublethal levels of ingestion. Sublethal doses of HCN may also be converted to thiocyanate, a well-known goitrogen. Although many food legumes contain only low levels of goitrogens, the conversion of cyanogens into goitrogens

(R)-Amygdalin CH2OH O OH

Linamarin H

C

H3C

N

CH2

O

O

CH2OH O OH

O

OH

HO OH

HO

CH3 C C

O

HO OH

OH

(R)-Lotaustralin

(R)-Prunasin H CH2OH O OH

C

H3C

N CH2OH O OH

O

C2H5 C

O

HO

HO

OH

OH

N

C

H

CH2OH CH2OH

O

OH HO

C

O OH HO

HO

O

O

H

C

C

N

OH

OH HO

(S)-Dhurrin

(R)-Taxiphyllin

(S)-Proteacin

C

H

N

C CH2OH O CH2OH O OH

O

HO OH

Figure 10.2 Chemical structures of some well-known cyanogenic glycosides.


N

O OH HO OH

C

N

C

C C

C

H2 C H2 C

C

C

C

CC

C

C

C

CH3

C Enzyme

CH3

Acid

C C

H2C

C

C

C

Hydroxynitrile lyase

CH3

CH

C

CH3

C C

C

β-D-Glucose

Linamarin

CC

C

H2 C

C

CC

C

C

CH3

CH3

Acetone cyanohydrin

Acetone

Figure 10.3 Enzymatic hydrolysis of linamarin, the cyanogenic glycoside of lima beans.

Amygdalin CH2OH O OH

H O

N

CH2

OH β-Glucosidase

O

OH OH

OH

HO

C O

HO

C

CH2OH O OH

H2O

HO OH

(R)-Mandelonitrile H H CH2OH O OH

C

N

OH

O

β-Glucosidase

HO

H2O

HO

O

OH HO OH

OH

N

C

CH2OH

C

C

Prunasin

Hydroxynitrile lyase

H O

HCN

C

Benzaldehyde

Figure 10.4 Stepwise hydrolysis of amygdalin, a cyanogenic glycoside found in bitter almonds, yielding prunasin, (R)-mandelonitrile, benzaldehyde, and HCN.


Table 10.14 Plants

Yield of HCN Released from Food

Plant

HCN yield, mg/100 g

Bitter almond Seeds Young leaves Wild cherry, leaves Apricot, seeds Peach Seeds Leaves Sorghum Whole plant, immature Mature seeds Etiolated shoot tips Young green leaves Bamboo Stem, unripe Tops of unripe sprouts Linseed (flax) Seedling tops Linseed cake Bitter cassava Leaves Inner part of tuber Dried root cortex Whole root Fresh root bark Fresh stem bark Vicia sativa, seeds Lima bean varieties Java, colored Puerto Rico, black Burma, white Arizona, colored America, white

290 20 90–360 60 160 125 250 0 240 60 300 800 910 50 104 33 245 53 89 133 52

seed, and cassava. Nearly 80 naturally occurring glucosinolates have been discovered (Verkerk et al., 1998). The common skeletal structure for glucosinolates that is now widely accepted is shown in Figure 10.5. It has a sulfonated oxime grouping, and the sugar in almost all cases is D-glucose. The glucosinolate side chain may comprise aliphatic (saturated and unsaturated), aromatic, or heteroaromatic groupings and commonly includes hydroxy groups (which may occasionally be glycosylated) and terminal methylthio groups and their oxidized analogs, esters and ketones (Fenwick et al., 1989; Verkerk, et al., 1998). It is the side chain that determines the chemical nature of the products of enzyme hydrolysis and, thereby, their biological effects and potencies. The chemical properties and occurrence of glucosinolates and their breakdown products have been reviewed extensively by Rosa and coworkers (1997). Several important glucosinolates in plants consumed by animals and humans are listed in Table 10.15. The total glucosinolate contents of agriculturally important plants are summarized in Table 10.16. The presence of glucosinolates in brassica plants is always associated with an enzyme system, myrosinase (also called thioglucosidase or thioglucoside glucohydrolase, EC 3.2.3.1). The enzyme is located in cellular compartments separate from glucosinolates in the plant and is released when plant cells are damaged by cutting or chewing (Fenwick and Heaney, 1983). The glucosinolate-myrosinase system may have several functions in the plant. These include plant defense against fungal diseases and

312 300 210 17 10

Glucosinolate R

S

C6H11O5

N

O

C

Source: Compiled from Poulton (1983) and Tewe and Iyayi (1989).

H2O

may be a factor in causing goiter in certain parts of the world.

SO3-

Thiglucosidase (Myrosinase)

SH R

C NOSO3-

D-Glucose

10.9 GLUCOSINOLATES Glucosinolates are secondary plant metabolites found exclusively in cruciferous plants. These sulfur-containing glycosides occur at highest concentrations in the families Resedaceae, Capparaceae, and Brassicaceae. They are found in such common vegetables as cabbage, turnips, rutabagas, mustard greens, horseradish, radish, mustard


R

N

C

S

Isothiocyanate

R

C

N

Nitrile

R

S

C

N

Thiocyanate

Figure 10.5 Enzymatic breakdown of glucosinolate.

Table 10.15 Nomenclature and Structure of Common Glucosinolates in Edible Plants

Table 10.16 Total Glucosinolate Content of Agriculturally Important Crops

Common name

Species

Aliphatic glucosinolates Glucoiberin Progoitrin Sinigrin Gluconapoleiferin Glucoraphanin Glucoalyssin Glucocapparin Glucobrassicanapin Glucocheirolin Glucoiberverin Gluconapin Indole glucosinolates 4-Hydroxyglucobrassicin Glucobrassicin 4-Methoxyglucobrassicin Neoglucobrassicin Aromatic glucosinolates Glucosinalbin Glucotropaeolin Gluconasturtiin

Structure 3-Methylsulphinylpropyl 2-Hydroxy-3-butenyl 2-Propenyl 2-Hydroxy-4-pentenyl 4-Methylsulphinylbutyl 5-Methylsulphinylpentyl Methyl 4-Pentenyl 3-Methylsulphonylpropyl 3-Methylthiopropyl 3-Butenyl 4-Hydroxy-3-indolylmethyl 3-Indolylmethyl 4-Methoxy-3-indolylmethyl 1-Methoxy-3-indolylmethyl p-Hydroxybenzyl Benzyl 2-Phenethyl

Cabbage White Red Savoy Chinese cabbage Brussels sprouts Cauliflower Calabrese Turnip Radish Oil Red White Horseradish Mustard White Black Rapeseed

Glucosinolate content, mg/g 0.26–1.56 0.41–1.09 0.47–1.24 0.17–1.36 0.60–3.90 0.61–1.14 0.42–0.95 0.21–2.27 0.92–1.12 0.42–1.17 0.57–1.19 33.2–35.4 22.0–52.0 18.0–60.0 13.0–42.0

Source: Compiled from Deshpande and Sathe (1991) and Verkerk et al. (1998).

Source: Compiled from Deshpande and Sathe (1991) and Verkerk et al. (1998).

pest infestation, sulfur and nitrogen metabolism, and growth regulation (Verkerk et al., 1998). Myrosinase has been found in the seed, leaf, stem, and roots of glucosinolate-containing plants, and the activity appears to be higher in the young tissues of the plant. Glucosinolates that act as progoitrins are the source of organic nitriles, isothiocyanates, and SCN ions. Some of these compounds have been shown to be quite harmful if consumed in sufficient amounts by humans and animals. They contribute to the flavor of all brassica plants. These compounds are usually formed by the hydrolysis of glucosinolates by myrosinases (Figure 10.5); the enzyme system, however, does not become active until the wet raw plant material is crushed. Less frequently, the aglucon products from the glucosinolates may be formed by chemical hydrolysis or by the action of enzymes from nonplant sources, such as intestinal microflora. The isothiocyanate ion formed upon enzymatic hydrolysis is unstable and cyclizes to yield 1,5-vinyl-2-thiooxazolidine, commonly known as goitrin. Goitrogens act primarily by preventing iodine uptake in the thyroid gland and, hence, impairing the synthesis of the thyroid hormones triiodothyronine and thyroxine. Goitrogens in cassava are also believed to be re-


sponsible for the differential distribution and severity of endemic goiter in some parts of Africa. The relative amounts of glucosinolates in a given species vary as a result of both genetic factors and agronomic practices. Similarly, any food processing method that causes disruption of cellular integrity causes at least a partial loss of glucosinolates by hydrolysis. Although heat processing inactivates myrosinases, some loss of glucosinolates may still occur through leaching into the cooking water. However, plant breeding remains an attractive alternative to achieve greater reduction in the glucosinolate content of cruciferous plants. In spite of their known toxicological effects, glucosinolates do seem to have certain beneficial health effects. The protective effect of cruciferous vegetables against cancer has been suggested to be due in part to the relatively high content of glucosinolates (Verkerk et al., 1998). This characteristic distinguishes them from other vegetables. Isothiocyanates that arise in plants as a result of enzymatic cleavage of glucosinolates by the endogenous enzyme myrosinase are attracting increasing attention as chemical and dietary protectors against cancer. These anticarcinogenic activities have been demonstrated in rodents (mice and rats) with a wide variety of chemical carcinogens (Table 10.17). The anticarcinogenic effects of isothiocyanates are attributed to a blocking effect involving the induction of

Table 10.17 Protection Against Chemical Carcinogenesis in Rat and Mouse Organs by a Variety of Isothiocyanates and Glucosinolates Protective isothiocyanates α-Naphthyl-NCS, β-naphthyl-NCS Phenyl-[CH2]n-NCS, where N = 0, 1, 2, 3, 4, 5, 6, 8, 10 PhCH(Ph)CH2-NCS, PhCH2CH(Ph)-NCS CH3[CH]n-NCS, where N = 5, 11 CH3[CH2]3CH(CH3)-NCS Sulforaphane, CH3S(O)[CH2]4-NCS 2-Acetylnorbornyl-NCS (three isomers) Protective glucosinolates Indolylmethyl glucosinolate (glucobrassicin) Benzyl glucosinolate (glucotropaeolin) 4-Hydroxybenzyl glucosinolate (glucosinalbin) Carcinogens employed 3′-Methyl-4′-dimethylaminoazobenzene 4-Dimethylaminoazobenzene N-2-fluorenylacetamide, acetylaminofluorene 7,12-Dimethylbenz[a]anthracene (DMBA) Benzo[a]pyrene Methylazoxymethanol acetate N-Nitrosodiethylamine 4-(Methylnitroamino)-1-(3-pyridyl)-1-butanone (NNK) N-Nitrosobenzylmethylamine (NBMA) N-Butyl-N-(4-hydroxybutyl)nitrosamine Tumor target organs Rat: Liver, lung, mammary gland, bladder, small intestine/colon, esophagus Mouse: Lung, forestomach Source: Compiled from Talalay and Zhang (1996) and Verkerk et al. (1998).

phase II enzymes in the small intestinal mucosa and liver (Zhang et al., 1992; Talalay and Zhang, 1996) and a suppressing effect involving suppression of tumor development by inducing programmed cell death, apoptosis (Smith et al., 1996). The evidence for anticarcinogenic effects of brassica vegetables in humans is strongly supported by evidence obtained with experimental animals. In a review by Steinmetz and Potter (1991), the overall conclusion from an analysis of 115 case-control studies was that a relatively high consumption of brassica vegetables was associated with a reduction in risk of cancer at many sites. For broccoli consumption in particular, there was a uniform protective effect, with no contrary evidence in any study. Consumption of brassicas, which might be expected to yield high levels of indoles and isothiocyanates, was particularly strongly associated with a lower risk of colon cancer. In view of this evidence concerning their beneficial biological activities in humans, factors inducing and di-


recting indole glucosinolate metabolism in plants need to be studied in much greater detail.

10.10 FAVIC AGENTS (FAVISM) The occurrence of sporadic cases of acute hemolysis after ingestion of broad beans (also known as fava beans, Vicia faba) was first recorded in the medical literature around the mid-1850s (Chevion et al., 1983). Since then several clinical reports as well as epidemiological, genetic, and biochemical studies have contributed to the characterization of this disease, termed favism, and to the elucidation of its cause and pathogenesis. Fava beans are commonly grown and consumed in large quantities in the Middle East, Far East, and North Africa. Except for the presence of vicine and convicine, the two β-glycosides implicated in the cause of favism and relatively low concentrations of other antinutrients common in legumes, fava beans are an excellent source of dietary protein. Vicine and convicine have also been reported in sweet lupine (Lupinus albus). One study, however, has clearly shown that these glycosides are only associated with the Vicia species and are not present in any other food plants (Pitz et al., 1980). These authors reported the average dry seed concentrations of vicine and convicine to be, respectively, 0.72% and 0.27% in V. faba minor (fava beans), 0.71% and 0.19% in V. faba major (broad beans), and 0.75% and 0.08% in V. narbonensis, a wild type of fava bean. Both environmental and genetic factors seem to affect the concentration of vicine and convicine in the seed of Vicia faba. Fresh green seeds contain much higher levels of glycosides than do mature seeds. The glycosides are synthesized very early in the development of the seed, and their concentration within the seed decreases with the increasing maturity of the seed. Vicine and convicine are hydrolyzed by intestinal microflora to highly reactive free radical–generating compounds, divicine and isouramil, respectively. The chemical structures of these four compounds are shown in Figure 10.6. Divicine and isouramil have been strongly implicated as the causative agents in favism. Their free radicals also cause other adverse effects, including lipid peroxidation, altered fat and mitochondrial metabolism, and possibly diabetes (Marquardt, 1989). Some of these adverse effects can be overcome by increasing the dietary intake of such antioxidants as vitamins A, C, and E, as well as chelating agents such as EDTA. Favism occurs only in individuals with a deficiency of glucose-6-phosphate dehydrogenase (G6PDH), an enzyme that has an important function in red blood cell metabolism. It is essential to maintain adequate levels of the

Vicine O

OH

O

O

HN N

H2N

Divicine

NH2

OH HOH2C O

OH

Convicine O

O

H2N

N

N H

OH

NH2

NH2

Isouramil O

O

HN

OH

HN

OH HOH2C O

OH

HN OH

O

N H

NH2

Figure 10.6 Structures of vicine and convicine. Their active aglycones, divicine and isouramil, respectively, arise from the removal of their sugar (glucose) by a β-glucosidase.

reduced form of glutathione (GSH) and NADPH. GSH and NADPH help to prevent oxidative damage to erythrocytes. The red blood cells of individuals with a deficiency of erythrocyte G6PDH are thus susceptible to oxidative damage. G6PDH-deficient individuals are subject to hemolytic crises from a variety of substances; among them is primaquine, an antimalarial drug. G6PDH deficiency is common in Mediterranean and Middle Eastern population groups and is an inborn error of metabolism. By nature, vicine and convicine, in which an autooxidizable hydroxyl group at C-5 of the pyrimidine moiety is blocked by the β-glycosidic bond, are inert. However, their corresponding aglycones, divicine and isouramil, rapidly lower the glutathione levels of erythrocytes deficient in G6PDH, causing hemolysis. The clinical manifestations of favism include hemolytic anemia, hemoglobinuria, and jaundice, often accompanied by high fever. It usually begins suddenly after the inhalation of pollen or a few hours after ingestion of the beans. In severe cases, death may occur in 24 to 48 hours. Fava beans also contain 3,4-dihydroxy-L-phenylalanine (L -DOPA) (Kosower and Kosower, 1967). DOPA may be transformed to dopaquinone by the action of tyrosinase. The quinone is active in causing a decrease in GSH in erythrocytes in G6PDH-deficient humans, whereas LDOPA is not (Beutler, 1970). Indeed, L-DOPA is present in garden pea (Pisum sativum), which rarely if ever, causes acute hemolysis as seen in favism. It is, however, possible that L-DOPA and divicine or isouramil may act synergistically in causing a lowering of erythrocyte GSH level in


these subjects (Razin et al., 1968). Ascorbic acid also seems to enhance the hemolytic property of divicine and isouramil. Favism is not known to occur in normal individuals and, hence, is not a serious problem. The only way of diminishing the risk of this disease in susceptible populations appears to be through effective genetic breeding. Since the glycosides appear to occur only in the cotyledon portions of the seed, dry milling of dehulled fava beans does not lower their concentration (Elkowicz and Sosulski, 1982). Wet-processing methods, on the contrary, remove over 97% of vicine and convicine. Cooking and sprouting of fava beans have a negligible effect on their total glycoside content; extracting whole or dehulled beans with 1% acetic acid or treatment with β-glucosidase has been shown to be highly effective (Hegazy and Marquardt, 1983; Arbid and Marquardt, 1985; Hussein et al., 1986).

10.11 LATHYROGENS (LATHYRISM) Consumption of the seeds of certain Lathyrus species results in a neurological disorder characterized by spastic paraparesis in humans and animals. This disease, lathyrism, is quite insidious and may be precipitated by fatigue and exposure to cold and wetness. The symptoms begin with back pain and weakness and stiffness of the legs. Muscular weakness, and in severe cases, paralysis of the legs follow, limiting the victim’s mobility to crawling. Males between 20 and 29 years old appear to be most sen-

sitive to lathyrogenic agents and are the ones usually affected most (Concon, 1988). Presently, lathyrism is confined mostly to the Indian subcontinent. Lathyrism has been known in humans at least since the days of Hippocrates (Seley, 1957) and still occurs in epidemic proportions in certain areas of the world during seasons of flood and drought, a time when other plants are destroyed and the species of L. sativus thrives and provides survival food (Roy and Spencer, 1989). The causative factor of this disease is still unknown. However, certain lathyrogens have been implicated and studied extensively. Two types of lathyrism are known: osteolathyrism and neurolathyrism. Osteolathyrism (also known as odoratism) is caused in rats and other animals by the ingestion of L. odoratus, L. hirsutus, and L. pusillus seeds; neurolathyrism is caused in humans by the prolonged consumption of seeds of L. sativus. A dipeptide (N-γ-glutamyl) aminopropionitrile was the first toxic principle to be detected in Lathyrus species. The β-aminopropionitrile (BAPN) (Figure 10.7) moiety of this dipeptide was shown to be responsible for osteolathyrism in experimental animals (DuPuy and Lee, 1954). BAPN, however, does not have any toxic effects on the nervous system. The human disorder is precipitated by excessive consumption of Lathyrus sp. seeds that contain the neurotoxin β-N-oxalylamino-L-alanine (BOAA) (Figure 10.7). Compounds possessing osteolathyrogenic potency can be broadly divided into four major groups: nitriles, urides, hydrazides, and hydrazines (Table 10.18). They show no structural relationship except that they all have, or

NC CH2

CH2

NH2

β-Aminopropionitrile (BAPN)

O HOOC C N CH2 H

CH COOH NH2

β-N-Oxalylamino-L-alanine (BOAA) Figure 10.7 Chemical structure of an osteolathyrogen β-aminopropionitrile (BAPN) and a neurolathyrogen β-N-oxalylamino-L-alanine (BOAA).


potentially have, an available terminal primary amine group (Barrow et al., 1974). Osteolathyrogens interfere with the initial reaction in the formation of cross-links in connective tissues. The results are increasing solubility of collagen, bone deformities, and modification of arterial elastin with consequent reduction in the tensile strength of aorta. Neurolathyrogens, such as BOAA, induce convulsions in rodents and primates, and in the rat, vacuolar degeneration of dendrites and cell bodies of cirumventricular organs (areas where the blood-brain barrier is normally absent), including the area postrema, subcommissural organ, subfornical organs, and arcuate-median eminence region of the hypothalamus (Olney et al., 1976). Because of its structural similarity to glutamic acid, BOAA also interferes with the high-affinity transport of both aspartate and glutamate and induces selective spinal cord abnormality. Similar neurolathyrogens have also been identified in the seeds of Vicia spp. The acutely neurotoxic amino acids from Lathyrus and Vicia species are summarized in Table 10.19.

Table 10.18

Compounds Having Osteolathyrogenic Potency

Organic nitriles β-Aminopropionitrile (BAPN) and β-(γ-glutamyl)aminopropionitrile Aminoacetonitrile (AAN) Methyleneaminonitrile (MANN) β,β′-Iminodipropionitrile (IDPN) β-Hydrazinopropionitrile 2-Cyanopropylamine (2-CPA) Ureides Semicarbazide (SCA) Acetone semicarbazide Hydrazides Nicotinic and isonicotinic acid hydrazide Cyanoacetic acid hydrazide Benzhydrazide Glutamic acid hydrazide γ-L-Glutamylhydrazide Carbohydrazide Thiohydrazide Hydrazines Hydrazine hydrate Phenyl hydrazine Symmetrical and unsymmetrical dimethyl hydrazine Miscellaneous Cyanide β-Mercaptoethylamine Thiosemicarbazide Source: Compiled from Barrow et al. (1974), Roy (1981), and Roy and Spencer (1989).

Table 10.19 Naturally Occurring Neurotoxic Amino Acids and Their Derivatives in Lathyrus and Vicia Species Toxic amino acid and derivative β-Cyano-L-alanine and γ-glutamyl-β-cyano-L-alanine L-α,γ-diaminobutyric acid, L-2,4-diaminobutyric acid, and γ-N-oxalyl derivative β-N-oxalyl-L-α,β-diaminopropionic acid (ODAP), or β-N-oxalylamino-L-alanine, L-3-oxalylamino-2-aminopropionic acid (OAP)

Source Vicia sativa, V. angustifolia Lathyrus latifolius, L. sylvestris, V. aurantica L. sativus, L. cicera, L. latifolius

Source: Compiled from Roy (1981) and Roy and Spencer (1989).

Several methods have been suggested to remove BOAA from L. sativus seeds for human consumption (Mohan et al., 1966). These include cooking the seeds in an excessive amount of water followed by draining, soaking the seeds in cold water overnight, and steeping the dehusked seeds in hot water. More BOAA is removed from dehusked than from whole seeds; complete removal is not possible. A maximum of 80% of BOAA can be removed from the seeds by any of the methods. Roasting the whole seed at 150°C for 20 minutes also destroys over 85% of BOAA. The levels of BOAA have also been reduced to minimal amounts by careful selection and plant breeding techniques; a safe level has not yet been defined.

10.12 TOXIC AMINO ACIDS In addition to the toxic lathyrogenic derivatives of amino acids, several other uncommon amino acids occur in the

Table 10.20

human food chain. These are usually the structural analogs of the protein amino acids, which exert their toxic effects as antimetabolites. They are usually found as free or simple condensation products in the form of γ-glutamyl, acetyl, methyl, and oxalyl derivatives. A summary of some of the more commonly occurring unusual amino acids in food plants is given in Table 10.20. Nonprotein toxic amino acids are considered secondary metabolites. Their functions in the plant are not fully understood, although several studies have indicated that they form a part of a chemical defense mechanism of the plant against predators (Roy and Spencer, 1989; Evans, 1983). Some of these compounds are toxic to specific microorganisms, insects, birds, and mammals. Most of those that are toxic to humans give rise to chronic, rather than acute, symptoms of poisoning. This suggests that low concentrations of a particular toxin can have a cumulative harmful effect if they constitute a part of the normal diet over a prolonged period. Toxic amino acids, which are

Some Toxic Amino Acids Commonly Occurring in Plant Foods and Forages

Amino acid N-(3-hydroxypyridone-4)-2-aminopropionic acid (mimosine) 2-Amino-4-(guanidinooxy)butyric acid (canavanine) L-2-Amino-6-amidinohexanoic acid (indospicine) 5-Hydroxy-L-tryptophan (5-HTP) 2-Amino-3-methylaminopropionic acid L-3,4-dihydroxyphenylalanine (L-DOPA) 3-Methylenecyclopropylpropionic acid (hypoglycin A) Seleno-amino acids (methylselenocysteine, selenocystathionine, selenocystine) 3-Cyanoalanine and 4-Glutamylcyanoalanine δ-Acetyl ornithine Homoserine and o-oxalylhomoserine Lathyrine, γ-methyl glutamic acid, γ-hydroxynorvaline, and γ-hydroxyhomoarginine Homoarginine and pipecolic acid Source: Compiled from Roy and Spencer (1989) and Evans (1983).


Source Many species of Mimosoidae (a subfamily of Leguminosae) Many species of Papilionoidae (a subfamily of Leguminosae) Tropical legume Indigofera spicata Griffonia simplicifolia Cycas spp. Vicia and some other legumes Plants of Sapindaeceae family Plants grown on selenium-rich soils Vicia spp. Lathyrus spp. Lathyrus spp. Lathyrus spp. Lathyrus and Vicia spp.

close structural analogs of protein amino acids, exert their toxicity by disrupting the enzyme systems. For example, the seleno amino acids are incorporated into enzymes in place of the normal sulfur amino acids, and canavanine replaces arginine in nuclear proteins. Mimosine is a potent inhibitor of cystathionine synthetase enzymes in the liver. Some uncommon amino acids also act as precursors of normal metabolites. Thus, 5-hydroxytryptophan gives rise to 5-hydroxytryptamine, a physiologically active amine in the mammalian brain. Others, such as L-DOPA, have been used in the treatment of Parkinson’s disease. As attempts are made to increase the food supply of the world by introducing plants not traditionally used for food and fodder, it is quite likely that the nonprotein amino acids may give rise to increased problems of toxicity in the future.

10.13 TOXIC FATTY ACIDS Either the normal fatty acids in foodstuffs that are of nutritional value are without exception saturated or unsaturated straight chains or they rarely exceed 20 carbon atoms in length. Furthermore, the double bonds in unsaturated fatty acids follow a specific pattern: position 9 in the monoenes, palmitoleic, and oleic acids; positions 9 and 12 in the diene, linoleic acid; positions 9, 12, and 15 in the triene, linolenic acid; and positions 5, 8, 11, and 14 in the tetraene, arachidonic acid. The specific structures of nutritional fatty acids suggest that any deviation from the norm may result in adverse effects, unless the animal system can successfully degrade the acid to nontoxic metabolites. Several unusual fatty acids have been shown to be toxic to humans. These include erucic acid, sterculic and malvalic acids (cyclopropene fatty acids), cetoleic acid, and in the presence of Refsum’s syndrome, phytanic acid (Concon, 1988). Cetoleic acid is of animal origin and is found in herring oil (Beare-Rogers et al., 1971). Its toxic effects are similar to those of erucic acid. Phytanic acid does not occur endogenously in plant foodstuffs. It is, however, a contaminant in ruminant fat and possibly dairy products (Hansen, 1965a, 1965b; Patton and Benson, 1966). Erucic acid and the cyclopropene fatty acids are the only ones in this group that are endogenous in plant foods. 10.13.1 Erucic Acid Erucic acid (C22:1, ω9) is found largely in the plant family Cruciferae, notably in Brassica spp. It accounts for as much as 20% to 55% of the fatty acid content of the oil derived from rapeseed (B. rapus and B. campestris) and mustard (B. hirta and B. juncea). As early as 1960, Roine and his collaborators reported myocardial effects in rats


fed 50% to 70% of their caloric intake as rapeseed oil. Weanling rats fed high levels of rapeseed oil experience accumulation of fat in the heart muscles even after the first day of feeding; the fat droplets are scattered throughout the myocardium. Subsequent studies have shown that even an intake of 4 energy percent in the form of erucic acid can lead to certain morphological defects (Engfeldt, 1975; Ziemlanski, 1977). The mechanism for the development of the abnormalities shown in experimental animals on erucic acid diets is thought to be a blockage of the β-oxidation of the fatty acids in mitochondria (Engfeldt, 1975) Rapeseed oil also interferes with the ability of the mitochondria in heart muscles to oxidize substrates such as glutamate and thus impairs the rate of ATP synthesis (Houtsmuller et al., 1970). This does not occur with liver mitochondria. Thus, the fatty accumulation in the heart muscle is due to the failure of the myocardium to oxidize erucic acid and/or convert it to oleic acid. High levels of erucic acid in the diet also impair the growth of chickens (Salmon, 1969a); ducklings, guinea pigs, hamsters, and mice (Thomasson et al., 1970); pigs (Thoron, 1969); and turkeys (Salmon, 1969b). The retardation of growth in rats also can be accomplished by feeding other fats mixed with erucic acid (Beare et al., 1959; Thomasson and Boldingh, 1955). Because a large intake of erucic acid is necessary to induce myocardial damage in animals, the hazard of erucic acid toxicity in humans is probably minimal. The worldwide production and increasing use of rapeseed and mustard seed oils in margarine and the important role they play in the farming systems of certain countries, such as Canada and India and in northern Europe, however, have called for intensive studies in these areas. Plant breeding efforts during the past four decades have resulted in the identification and development of low erucic acid–content rapeseed varieties. 10.13.2 Cyclopropene Fatty Acids The oils or fat of every plant of the order Malvales that have been examined thus far, with one exception, contain cyclopropene fatty acids (Eckey, 1954; Phelps et al., 1965). The exception is cocoa butter from Theobroma cocoa (Hilditch and Williams, 1964). Two important acids in this group are sterculic (19 carbons) and malvalic (18 carbons) acids (Figure 10.8). From the food toxicological viewpoint, only the cyclopropene fatty acids in the oils of cottonseeds (Gossypium hirsutum) are of significance. Crude cottonseed oil may contain from 0.6% to 1.2% cyclopropene fatty acids in the form of sterculic and malvalic acids (Bailey et al., 1966). Processing may reduce these levels by 0.1% to

Table 10.21

H2 C

Saponin Content of Staple Plant Foods

Plant CH3(CH2)7C

C

(CH2)n

Malvalic acid:

n=6

Sterculic acid:

n=7

COOH

Figure 10.8 Cyclopropene fatty acids found in cottonseed oil.

0.5%. The cottonseed meal may contain about 0.01%, depending on the quantity of residual oil. Hydrogenation probably destroys some of the biological effects of these acids (Masson et al., 1957). Both sterculic and malvalic acids have been shown to possess carcinogenic activities. In their presence, the carcinogenic activity of aflatoxin B1 is also considerably enhanced (Lee, 1967; Sinnhuber et al., 1966). Sterculic acid also prevents the conversion of exogenous stearic acid to oleic acid. This results in increased levels of saturated fatty acids in body fat at the expense of monounsaturated fatty acids (Johnson et al., 1967). This increase in saturated fatty acids in body fat cannot be overcome by the addition of oleic or linoleic acids in the diet (Evans et al., 1963). One possible mechanism suggested is the inhibition of stearate desaturase by the cyclopropene group in these fatty acids (Johnson et al., 1967; Raju and Reiser, 1969). The importance of cyclopropene fatty acids to human health is unknown. Cottonseed oil has been used for several years in food preparation, apparently with no ill effects (Concon, 1988). However, experimental or epidemiological evidence to this effect is lacking. The carcinogenicity of these fatty acids together with their biological and biochemical effects give them greater toxicological significance than hitherto realized.

10.14 SAPONINS Saponins are a complex and chemically diverse group of compounds found primarily in many plants as well as in a number of marine animals. Their physiological effects are also as diverse as their chemical structures and properties, and not all of them are toxic. Saponins, however, occur in significant amounts in many commonly used food and forage plants (Table 10.21). Although many of these are herbs, spices, or medicinal plants and are consumed only in small amounts, some are staple items of the diet of a


Chickpea (Cicer arietinum) Soybean (Glycine max) Dry bean (Phaseolus vulgaris) Mung bean (Phaseolus aureus) Broad bean (Vicia faba) Lentil (Lens culinaris) Green pea (Pisum sativum) Peanut (Arachis hypogaea) Asparagus (Asparagus officinalis) Spinach (Spinacea oleracea) Silver beet (Beta vulgaris) Sesame seed (Sesamum indicum) Oat (Avena sativa)

Saponin content, g/kg 2.3–60 5.6–56 4.5–21 0.5–5.7 3.5 1.1–5.1 1.1–1.8 0–16 15 47 58 3 1–1.3

Source: Compiled from Price et al. (1986), Fenwick and Oakenfull (1981), Curl et al. (1985), and Oakenfull and Sidhu (1989).

large part of the world population. Among the major ones are chickpeas in the Middle East and the Indian subcontinent, soybeans in much of Southeast Asia, and peanuts in central Africa. The major forage plants containing saponins include alfalfa, cloves, guar, sunflower, and lupine. Chemically, saponins have a triterpene or steroid backbone linked to one or more sugar groups (Price et al., 1987). There are thus two broad subdivisions of saponins: those with triterpenoid aglycones and those with steroid aglycones. Almost all the saponins in food and forage plants are of the triterpene class. The chemical structures of two well-characterized and biologically active saponins are shown in Figure 10.9. Within the same plant species, the saponins differ in composition as well as in quantity. For example, DuPuits and Lahontan alfalfa varieties contain 25 different saponins, but the chemical composition of the saponins in the two varieties is not the same (Birk and Peri, 1980). The aglycones of DuPuits saponins are mainly of the medicagenic acid type, whereas the saponins of Lahontan contain soybean sapogenins and monocarboxylic acids as aglycones. Also, DuPuits saponins lack D -galactose, which is present in Lahontan saponins. The composition of alfalfa seed saponins seems to be similar to that of soybean seeds (Birk and Peri, 1980). Saponins are commonly isolated by extraction of the plant material with hot water or ethanol. On complete hydrolysis, they yield sapogenins, which are the aglycones of steroids (C27) or triterpenes (C30). Among food plants, soy saponins are the most thoroughly studied saponins. They were first identified and characterized in 1964 (Willner et al., 1964). The sugars present on the intact soy saponins

H3C

Medicagenic acid 3-β-O-triglucoside CH2OH O OH

O

CH2 H3C

O

HO

OH OH

CH3

CH3

H

COOH

HO

O

HO

CH2OH O

OH

H

CH3

O H 3C

HO OH

H3C

COOH

CH3

Hederagenin H3C

CH3

COOH

HO CH3 HO H3C

CH2OH

Figure 10.9 Structural formula of the alfalfa saponin medicagenic acid 3-β-O-triglucoside, and the aglycone hederagenin of soyasapogenol.

include galactose, arabinose, rhamnose, glucuronic acid, xylose, and glucose. Despite their diverse chemical properties, saponins have a number of common and characteristic properties, including the following: 1. 2. 3. 4. 5.

Bitter taste Formation of stable foams in aqueous solutions Hemolysis of red blood cells Toxicity to cold-blooded animals, such as fish, snails, and insects Ability to interact with bile acids, cholesterol, or other 3-β-hydroxy steroids in aqueous or alcoholic solutions to form mixed micelles or coprecipitates

Saponins have had many industrial and commercial applications, including use as emulsifiers in soft drinks, shampoo, fire extinguishers, soap, and the synthesis of steroid hormones (Deshpande and Sathe, 1991). The wide range of chemical and physical properties of saponins is also reflected in the range of their physiological and pharmacological effects. Although they are extraordinarily physiologically active compounds, their reputation for toxicity is in general unfounded. Ingested saponins mostly remain


within the GI tract, and it is only when they enter the bloodstream that their hemolytic activity causes damage (Oakenful and Sidhu, 1989). Their physiological activity appears to arise from two main causes: their strong physical interactions as surface-active agents with dietary and intestinal components and their ability to interact with the membranes of the mucosal cells. Among the effects of saponins on animals are growth inhibition in swine and poultry, reduced palatability of food, and increased excretion of cholesterol. Alfalfa protein concentrate (APC) prepared from low-saponin varieties yields better growth performance in rats than the one prepared from the high-saponin variety (Hegsted and Linkswiler, 1980). The reduced weight gains with high-saponin APC were attributed to lower feed intake and lower protein digestibility. In recent years, the ability of saponin-rich foods to lower plasma cholesterol concentration and thus the risk of cardiovascular diseases has attracted much attention. Kritchevsky and colleagues (1975) were among the first to suggest that the saponins in alfalfa were the cause of the lower plasma cholesterol levels in rats fed alfalfa-based diets. Malinow and coworkers (1985, 1987) have since provided conclusive evidence that the saponins in alfalfa are indeed responsible for the lower plasma cholesterol con-

centrations and that alfalfa fiber in the absence of saponins is not responsible for this response. The only reported experiment in which human subjects were given saponins directly is that described by Bingham and associates (1978), who gave a group of 174 arthritic patients tablets of a saponin-rich extract from Yucca schidigen in a treatment that included controlled diets, exercise, and physiotherapy. A control group was given a placebo, with the same treatment. Substantial reductions in plasma cholesterol concentration were observed in response to the saponins, particularly in individuals who had initially higher levels. There were also significant reductions in plasma triglyceride concentration and blood pressure. Foods containing saponins as a significant, but naturally occurring component have also been shown to be effective in lowering plasma cholesterol concentration in hyperlipidemic humans. Mathur (1968) found that giving hypercholesterolemic patients chickpeas as an isocaloric substitute for wheat flour and other cereals lowered their mean plasma cholesterol concentration from 206 mg/100 ml to 160 mg/100 ml. Other legumes have been found to be similarly effective (Effect of legume seeds, 1980). The mechanism of this effect seems to be, at least in part, that saponins inhibit absorption from the small intestine of cholesterol or bile acids. Bile acid and cholesterol metabolism are closely interrelated, since the former are synthesized from cholesterol in the liver. Both are secreted by the liver as bile to promote the digestion and absorption of lipids. Bile acids are normally very efficiently recycled. The ability of some saponins to form mixed micelles with bile acids interrupts this cycle. Bile acids are not reabsorbed from such micelles and consequently are excreted. Similarly, the formation of molecular complexes of saponins with cholesterol prevents its absorption. The net result is a higher loss by fecal excretion of cholesterol and bile acids than can be compensated from endogenous or exogenous sources, with consequent reduction in the cholesterol levels of blood plasma, liver, and other tissues. Further elucidation of the chemical structures of various saponins and their interactions with cellular and membranal components will undoubtedly lead to a better understanding of the role of saponins in human nutrition and medicine. It also appears quite likely that in the coming years, saponins may actually be deleted from the antinutritional factors in human nutrition.

10.15 POTATO GLYCOALKALOIDS Steroidal alkaloids are commonly found in Solanum spp., including potato and tomato. Solanine, a glycoside containing a steroid alkaloid nucleus (aglycone) with a side


chain of sugars, was first discovered in potatoes in 1826 (Cheeke and Shull, 1985). The aglycone is called solanidine. Later on another glycoalkaloid, chaconine, was also discovered in potatoes. Solanine and chaconine have the same aglycone (solanidine) but differ with respect to the carbohydrate side chain (Figure 10.10). Since these early discoveries, several other alkaloids have been identified in potatoes. These were found to contain two different steroidal skeletons. Solanidane (solanidine type) contains the indolizidine system exemplified by solanines, chaconines, solanidine, leptines, leptinines, and demissidine. The second group, spirosolane (solasodine type), possesses oxa-azaspirodecane structure as represented by tomatidenol, and α- and β-solamarines (Jadhav et al., 1997). The hydrolysis products and sugar moieties of the glycoalkaloids are summarized in Table 10.22. Glycoalkaloids are toxic and thus are believed to be involved in pest resistance. These toxicants occur in potato tubers, peels, sprouts, and blossoms, and their concentration in tubers depends on cultivar, maturity, environmental factors, and stress conditions. Concentrations may vary because of fungal or bacterial infection and usually increase in response to wounding, apparently as a defense mechanism against potential disease. Glycoalkaloids, therefore, may function as phytoalexins (Jadhav et al., 1997). Green sprouts and green potato skins (peels) contain the highest concentrations of solanum alkaloids. The greening of potatoes occurs when the tubers are exposed to sunlight during growth or after harvest. The greening is due to chlorophyll; the increased concentration of solanum alkaloids in green potatoes occurs because similar environmental conditions promote the synthesis of both chlorophyll and glycoalkaloids (Jadhav et al., 1981). The two principal effects of solanum alkaloid poisoning are gastrointestinal tract irritation and impairment of the nervous system. Because of their relatively low gastrointestinal absorption, these alkaloids are much more toxic when administered parenterally then orally (Nishie et al., 1971). The glycosides are more toxic than the corresponding aglycones. Apathy, drowsiness, salivation, dyspnea, trembling, weakness, paralysis, and loss of consciousness are manifestations of the effects on the nervous system. Solanum alkaloids are cholinesterase inhibitors (Jadhav et al., 1981). Inhibition of this enzyme results in increased accumulation of acetylcholine in nerve tissue, thus impairing the neural function. This is manifested in such neurological signs as ataxia, convulsions, coma, muscle weakness, and involuntary urination. Gastrointestinal tract effects of solanum alkaloids include inflammation of intestinal mucosa, hemorrhage or

α-Solanine

CH3 CH3 N CH3 H

CH3 H CH2OH O HO CH2OH O OH

O

HO

H

H

H

O

O HO

O

OH

CH3 OH

OH

α-Chaconine CH3 CH3 N CH3 H

CH3 H HO

CH2OH O OH

O CH3 OH

O OH

H

H

H

O

O HO

O CH3 OH

Figure 10.10

OH

Molecular structures of potato glycoalkaloids.

ulceration, abdominal pain, constipation, and diarrhea. Teratogenic effects of potato alkaloids have also been suggested (Renwick, 1972). In addition to human poisoning, incidences of livestock poisoning have also been known in animals fed potato sprouts, peelings, and sunburned or spoiled potatoes. Potato vines have also caused toxicity, since the alkaloids are in highest concentration in green tissues.


Solanum alkaloids are not destroyed by boiling, baking, frying, or drying at high temperatures (Jadhav et al., 1997; Cheeke and Shull, 1985). The relatively rare occurrence of solanine poisoning has been mainly due to three factors: Solanine is poorly absorbed, it is hydrolyzed to a considerable extent in the gut to the less toxic solanidine, and urinary and fecal excretion of its metabolites is rapid. Nevertheless, new potato varieties are now routinely

Table 10.22 Glycoalkaloids and Their Hydrolysis Products (Aglycone and Sugar Moiety)a Glycoalkaloid α-Solanine

Aglycone

Sugar moiety

Solanidine

D-Glu

-D-Gal< β-Solanine γ-Solanine α-Chaconine

Solanidine Solanidine Solanidine

β-Chaconine γ-Chaconine α-Solamarine

Solanidine Solanidine Tomatidenol

β-Solamarine

Tomatidenol

Leptine I

O(23)Acetylleptinidine

Leptine II Leptinine I

O(23)Acetylleptinidine Leptinidine

Leptinine II

Leptinidine

Commersonine

Demissidine

L-Rha -D-Gal-D-Glu -D-Gal L-Rha -D-Glu< L-Rha -D-Glu-L-Rha -D-Glu D-Glu -D-Gal< L-Rha L-Rha -D-Glu< L-Rha L-Rha -D-Glu< L-Rha D-Glu -D-Glu< L-Rha L-Rha -D-Glu< L-Rha D-Glu -D-Glu< L-Rha D-Glu

-D-Gal-D-Glu< D-Glu

Demissine

Demissidine

D-Glu

-D-Gal-D-Glu< D-Xyl a

Glu, glucose; Gal, galactose; Rha, rhamnose; Xyl, xylose.

screened before release, and glycoalkaloid levels must be below 20 mg/100 g (Jadhav et al., 1997; Plhak and Sporns, 1997). Levels of 14 mg/100 g are bitter; levels above 20 mg cause a burning sensation in the mouth and throat.

10.16 OXALATES Certain families and species of plants contain relatively large amounts of oxalic acid, mainly as the soluble sodium or potassium salts or the insoluble calcium salts. Some of the common plant foods containing appreciable amounts of oxalates (fresh weight basis) are spinach (0.3% to


1.2%), rhubarb (0.2% to 1.3%), beet leaves (0.3% to 0.9%), tea (0.3% to 2.0%), and cocoa (0.5% to 0.9%). Lettuce, celery, cabbage, cauliflower, turnips, carrots, potatoes, peas, and beans also contain lesser amounts of oxalates. It is well known that humans excrete varying amounts of calcium oxalate crystals in the urine (6 to 45 mg/day, with a mean of about 20 mg/day in normal subjects). About two thirds of the urinary oxalate is derived from ascorbic acid and the amino acid glycine; the rest is from dietary oxalate and possibly from precursors, such as glycolic and glyoxylic acids. Like phytates, oxalates can decrease the availability of dietary essential minerals such as calcium. The adverse effects of oxalates in relation to calcium must be considered in terms of the oxalate/calcium ratio. On a milliequivalent basis, foods having a ratio greater than 1 may have serious effects on calcium availability (Table 10.23). Those with a ratio of 1 or below cause no difficulty in the availability of calcium as far as other calcium sources are concerned. In contrast to their role in calcium absorption, oxalates appear not to interfere in zinc absorption in zincdeficient rats (Welch et al., 1977). Somehow, a counteracting or protective mechanism may prevent the precipitation of zinc by oxalates. A metabolic disorder resulting in the production of renal stones and deposits of calcium oxalate in other tissues is often attributed to the excessive consumption of oxalates from foods. Under such conditions, urinary levels of oxalates and glycolates are greatly increased. This disorder is attributed to an inability to transaminate glyoxylate from glycine; this in turn results in increased formation of glyoxylate from glycolates and hence in oxalic acid (Fassett, 1973). However, the role of exogenous oxalates in the production of renal stones seems to be doubtful in most cases. Acute oxalate toxicity in humans is associated with corrosive gastroenteritis, shock, convulsions, low plasma calcium levels with correspondingly higher levels of oxalates, and renal damage. Acute oxalate poisoning in humans, however, is rare. Even for noticeable chronic toxic effects to occur, a rather improbable combination of circumstances (a very high intake of oxalate-containing foods plus a simultaneously low calcium and vitamin D intake over a prolonged period) is needed. In this connection, fatal rhubarb poisonings, which were thought to be due to the oxalate content, are probably caused by other factors even though the leaves may contain up to 1.1% oxalates (von Streicher, 1964). Also, the characteristic symptoms of oxalate poisoning, such as corrosive gastroenteritis, were absent in these fatal cases (Kalliala and Kauste, 1964; Tallquist and Vaananen, 1960).

Table 10.23

Foods Having an Oxalate/Calcium Ratio (MEQ/MEQ) > 1

Food

mg Oxalate/100 g

Average

Oxalate/CA2+

275–1336 270–730 300–700 320–1260 300–920 121–450 910–1679 500–900 50–150 20–141 300–2000 890 1100 300–1500 1586 1087

805 500 500 970 610 275 1294 700 100 80 1150

8.5 5.6 5.0 4.3 2.5 5.1 4.6 2.6 3.9 1.6 1.1 3.9 4.9 4.0 1.2 1.4

Rhubarb (Rheum rhaponticum) Common sorrel (Rumex acetosa) Garden sorrel (Rumex patientia) Spinach (Spinacea oleracea) Beet, leaves (Beta vulgaris, var. cicla) Beet, roots Purslane (Portulaca oleracea) Cocoa (Theobroma cocoa) Coffee (Coffea arabica) Potato (Solanum turberosum) Tea (Thea sinensis) New Zealand spinach (Tetragonia expansia) Pig spinach (Chenopodium spp.) Orache (Atriplex hortensis) Amaranth (Amarantus polygonoicles) Amaranth (Amarantus tricolor)

900

Source: Compiled from Gontzea and Sutzescu (1968) and Concon (1988). MEQ, milliequivalents.

It has been postulated that the anthraquinone glycosides may be responsible for fatal rhubarb poisonings (Von Streicher, 1964). Fresh rhubarb leaves may contain 0.5% to 1% anthraquinones; 10 to 20 g of fresh leaves has caused immediate poisoning in human volunteers (Schmid, 1951). Harmful oxalates in food may be removed by soaking in water. Consumption of calcium-rich foods, such as dairy products and seafood, as well as augmented cholecalciferol intake, are recommended when large amounts of highoxalate food are eaten (Schmid, 1951; von Streicher, 1964).

probably gave animals, including humans, the capability to detoxify these compounds very easily (Singleton and Kratzer, 1973). The first group includes phenolic acids such as caffeic, ferulic, sinapic, and gallic acids; their derivatives; flavonoids; lignin; hydrolizable and condensed tannins; and ellagic acids and their derivatives. The antinutritional effects of tannins were described earlier. Examples of the second group include gossypol, phlorizin, coumarins, myristicin, urushiols, and phenolic amines or catecholamines. Many of these compounds have specific pharmacological effects.

10.17 TOXIC PLANT PHENOLS AND ALCOHOLS

10.17.1 Gossypol

On the basis of frequency of occurrence, similarity in structure, and relative toxicity, phenolic compounds may be divided into two major groups: those having widespread and common occurrence in plant-derived foods and beverages, of which approximately 25 compounds have been identified, and more heterogenous groups of compounds, including a few dozen phenolic derivatives that are highly toxic or have potent pharmacological activities (Singleton and Kratzer, 1969). In the first group, although some compounds occur in relatively high concentrations in some foods, the majority are present in trace quantities. Because of the widespread consumption of these foods, it is presumed that these substances normally are devoid of acute toxicity at the levels usually found in food. Evolutionary adaptation


The polyphenolic gossypol pigments are primarily confined to the genus Gossypium and a few other members of the order Malvales. The toxicity of cottonseed meal is associated primarily with its gossypol content. Although cottonseed is a by-product of the cotton fiber, the processing of cottonseed is a major industry in the cotton-producing areas of the world. Of the various products (oil, meal, linters, and hulls) obtained from cottonseed, oil is the most valuable. Earlier, cottonseed meal was used principally as a protein supplement for ruminant livestock. The naturally occurring gossypol pigments of the cottonseed are recognized toxicants to monogastric animals, and this property has limited the use of cottonseed meals in feeds for swine and poultry. However, it also represents a potentially excellent source of low-cost, high-quality protein products for food. The refined cottonseed flour may contain up to

60% protein and can become a valuable and abundant source of supplementary protein in human nutrition. Thus, the presence of gossypol in the meal is of toxicological importance. Concerted research efforts have led to glandless, gossypol-free cottonseed varieties. Gossypol was first isolated in a crude form in 1861, but it was not until 1915 that its toxic properties were generally recognized (Abou-Donia, 1989). In addition to gossypol, cottonseed contains several cyclopropenoid fatty acids, sterculate, and malvalate. These are, however, a component of cottonseed oil rather than the protein fraction of the seed. There are at least 15 gossypol pigments or derivatives in extracts of cottonseed oils and meals, but only about 8 have been isolated in more or less purified form and characterized (Concon, 1988; Deshpande and Sathe, 1991). They include gossypol (yellow), diaminogossypol (yellow), 6-methoxygossypol (yellow), 6,6′-dimethoxygossypol (yellow), gossypurpurin (purple), gossyfulvin (orange), gossycaerulin (blue), and gossyverdurin (green). Gossypol occurs in a greater amount in raw cottonseed than in cottonseed that has been subjected to moist heat treatment during processing. In contrast, gossypurpurin and gossyfulvin occur in the cooked seed. Gossycaerulin occurs almost exclusively in cooked cottonseed. Gossypol is converted to gossypurpurin during maturation and prolonged storage of the seed (Berardi and Goldblatt, 1980). The structure for gossypol suggested by Adams and colleagues (1938) is now universally accepted. It is a binaphthyl compound containing a number of hydroxyl and formyl groups. These groups interact to form aldehyde (I), hemiacetal (II), and enolic quinoid (III) types of compounds to yield three tautomeric forms of gossypol (Figure 10.11). Practially, all gossypol in cottonseed plant occurs in pigmented glands whose liquid portion contains 20% to 46% gossypol. It is primarily synthesized in the roots. In glandless varieties, the transport of gossypol from the roots to the seeds is prevented; thus, they contain little or no gossypol. Gossypol has a wide range of toxicological, pharmacological, physiological, and biochemical effects. It is more toxic to nonruminants than to ruminants, thus limiting the use of cottonseed protein to humans and animals. Swine, guinea pigs, and rabbits are the most sensitive to gossypol; rats are the least. The toxic effects of gossypol in monogastric animals have been classified in three levels. Acute doses cause circulation failure; subacute doses, pulmonary edema. Chronic toxicity results in symptoms of ill health and malnutrition. Most of these toxic effects occur by nonspecific interaction with some dietary and body components, such as iron and protein, and/or specific inhibition of certain enzymes (Abou-Donia, 1989). Gossypol


has also caused reversible male antifertility in rats, hamsters, monkeys, and humans. From a nutritional viewpoint, the reaction of carbonyl groups of gossypol with the amino groups of the amino acids and proteins to form Schiff’s base–type derivative is significant. It renders the amino acids, particularly lysine via its ε-amino group, unavailable, thus lowering the nutritional value of the diet. The phenolic groups of gossypol may also combine with protein reversibly by hydrogen bonding and irreversibly by oxidation to quinines, which react with proteins. Several approaches have been used to reduce the gossypol content of cottonseed meals. One of the oldest techniques still used is controlled heating with moisture to react the gossypol with other seed components, especially lysine. The bound gossypol is less toxic. The toxicity of gossypol can also be reduced to varying degrees by adding metal ions, mainly iron, or chemically combining it with aniline, ammonia, and boric acid. The addition of large amounts of iron salts to cottonseed meal decreases the absorption of gossypol from the intestinal tract. The pigmented glands can be separated from the meal by a flotation procedure by using organic solvents. Gossypol exhibits a dose response, thus allowing the safe use of cottonseed meal in animal feedstuffs. FDA regulations specify that cottonseed products intended for human use in the United States contain no more than 0.045% free gossypol, and the Protein Advisory Group of the United Nations has set limits of 0.06% of free gossypol and 1.2% of total gossypol for human consumption in their program (Deshpande and Sathe, 1991). The widespread use of glandless cottonseed varieties, however, may render gossypol toxicity a subject of historical interest only. 10.17.2 Alkylated Catechols and Related Phenols Tropical plants of the family Anacardiaceae, which includes mango (Mangifera indica), cashew (Anacardium occidentale), and pistachio (Pistacia vera), contain a mixture of alkylated catechols that differ in the degree of unsaturation of the hydrocarbon substituent (Concon, 1988). Mango dermatitis is very similar to poison ivy. Although the poisonous compounds in mango have not been formally identified, they are presumed to be catechol derivatives. The active principle is located in the bark, stem, leaves, and peel of the fruit. The pulp itself is safe. Mango dermatitis is similar to other forms of what Merrill (1944) has called anacardiaceous dermatitis. For those who peel the fruit before eating it, mango dermatitis is manifested by acute erythematovesicular eruptions with some swelling in the lips, cheeks, chin, and sometimes

CHO

OH

OH

CHO

HO

A

OH

HO

OH

CH3 H3C H3C

CH3

HOHC

H3C

O

O

CH3

CHOH

HO

B

OH

HO

OH

CH3 H3C H3C

CH3

HOHC

H3C

OH

OH

CH3

CHOH

O

C

O

CH3 H3C

HO H3C

Figure 10.11

CH3

OH H3C

CH3

Three tautomeric forms of gossypol found in cottonseed meals: A, aldehyde; B, hemiacetal; C, quinoid.

hands. Sometimes stomatitis and acute gastrointestinal disturbance may also occur. The reaction generally appears within 6 to 24 hours after exposure, beginning with smarting, erythema followed by an intense burning sensation, and itching accompanied by vesiculation and sometimes edema. Adverse reactions to the toxic principle of mangoes appear to be dependent on individual susceptibility. 10.17.3 Cycasin and Related Azoxyglycosides The species of the plant family Cycadaceae thrive in the tropical and subtropical regions of the Pacific and Carib-


bean islands, Mexico, Florida, Japan, and Southeast Asia. They are extremely hardy and are able to withstand adverse climatic conditions. When most other food crops are destroyed, for example, during the typhoon season, they become the principal source of subsistence. Although there are nine genera in Cycadaceae, at least four species from three genera, viz., Cycas circinalis, C. revoluta, Encepharlartos barkeri, and Macrozamia spiralis, have been found to contain toxic compounds. The toxic principles from cycads have been identified, isolated, and crystallized. These differed only in the sugar moieties, having the same aglycone. Cycasin has been isolated from C. circinalis and C. revoluta. The latter

species also yields several other azoxyglycosides, called neocycasins. These have the identical aglycone to cyacasin (Nishida, 1959; Nagahama, 1964). Macrozamin has been crystallized from M. spiralis. Cycasin and macrozamin differ only in that the former contains glucose and the latter primeverose (6-[β-D-xylosido]-D-glucose) (Figure 10.12). The azoxyglycosides of cycads are harmless when administered parenterally, and only when they are taken orally are their toxic effects evident (Nishida et al., 1956). Both malignant and benign tumors of the liver, duodenum, colon, rectum, and kidneys are induced. The toxicity of the glycosides is dependent on their hydrolysis in the GI tract to the aglycone form. The carcinogenicity of cycasin or cycad meal has been demonstrated in mice, hamsters, and guinea pigs. They are also probable human carcinogens (Concon, 1988). The toxic compounds in cycads are highly watersoluble. The powdered or grated endosperm can be rendered nontoxic by washing and thorough soaking (Whiting, 1963).

Cycasin CH3N

N

CH2

CH2OH

O

O OH

O

OH OH

Macrozamin CH3N

N

CH2

CH2

O

O

O

OH

O

OH

OH O

OH

OH OH

Figure 10.12 Toxic azoxyglycosides found in cycads. Cycasin contains glucose; macrozamin has primeverose (6-[β-Dxylosido]-D-glucose).

10.17.4 Safrole Safrole (4-allyl-1,2-methylenedioxybenzene) is widely distributed in the plant kingdom (over 50 species and varieties representing some 10 plant families). This phenolic compound commonly occurs in many spices and such essential oils as star anise oil, camphor oil, and mace, ginger, and cinnamon leaf oil. Nutmeg is one of the best sources of safrole (Belitz and Grosch, 1987). Safrole was widely used as a flavoring agent in soft drinks and other food products. However, its use for these purposes was discontinued after its hepatocarcinogenic activity in rodents was discovered. The chemical structures of safrole and its three structurally related analogs, isosafrole, dihydrosafrole, and estragole, are shown in Figure 10.13. The LD50 values in rats for safrole, isosafrole, and dihydrosafrole are, respectively, 1950, 1340, and 2260 mg/kg body weight. The carcinogenic properties of safrole were established by the induction of liver tumors in rats fed high levels of the compound for 2 years. Malignant liver tumors develop at high safrole concentration (0.5% level in the diet); at lower levels (0.1%) benign tumors are developed. The three safrole derivatives, however, are not of equal potency (Kinghorn, 1983). In rats, safrole and isosafrole appear to be hepatocarcinogens; dihydrosafrole is carcinogenic for the esophagus. However, the high levels at which these compounds are carcinogenic suggest that they pose very little, if any, health hazard under normal dietary conditions in human nutrition.


O

O O

CH2

CH

O

CH2

Safrole

CH

CH

CH3

Isosafrole

O

OCH3 O

CH2

CH2

CH3

Dihydrosafrole

CH2

CH

CH2

Estragole

Figure 10.13 Chemical structures of safrole and its derivatives.

CH2

O

O

H3CO

CH2

O

O

H3CO

OCH3 CH2CH

CH2

Myristicin

CH2CH

CH2

O

Apiol

O

H3CO

CH2

H3CO CH2CH

CH2

Dill apiol O

CH2

O

H2C

O

O

O

O CH2 O

Sesamol

O

OH

Sesamin

O H2C O

O

O CH2

O

Sesamolin

O

O

CH2

O

CH2

O

O

O

Piperine H C H

C

C

H

O

C

C

Chavicine

N

H

C

C

C

C

C

H

H

H

H

O

N

CH2

O

O

Piperettine C H

Figure 10.14

C

H

H

H C H

C

C

C

H

Carcinogenic methylenedioxybenzenes from various foods.


C O

N

Several other methylenedioxybenzenes are found in many food sources. These include myristicin and related compounds found in nutmeg and other spices; apiol, found in oleoresin of parsley (Petroselinum crispum and P. sativum) and celery (Apium graveoleus); sesamol, sesamolin, and sesamin in sesame (Sesamum indicum) seed oil; and alkaloids of black pepper (Piper nigrum), piperine, chavicine, and piperettine (Figure 10.14). These all have structures very similar to that of safrole. However, Miller (1983) has shown that in male B6C3F1 mice, myristicin, dill apiol, and parsley apiol did not produce a significant incidence of tumors after 13 months. It is quite likely that their activity may be weakened by the methoxy substituents meta and/or ortho to the allyl group. Tests using higher doses on the same or different stains of animal may reveal their carcinogenic potential. The potential carcinogenicity of black pepper has been reported by Concon and associates (1979, 1981). In addition, the tumorigenicity of sesamol has been evaluated by Ambrose and colleagues (1958), who reported that an increased incidence of benign proliferative lesions is observed in rats fed for several months a diet containing sesamol.

Table 10.24

10.18 VASOACTIVE (PRESSOR) AMINES Some foods contain substantial enough quantities of toxic substances to induce noticeable changes in the cardiovascular system (vasoactive) or in mental function (psychoactive). The number of substances in food that produce alterations in cardiovascular function is, in reality, quite small. Some of the known vasoactive or pressor amines found in plants are listed in Table 10.24. The amounts of amines listed may appear minimal. However, these compounds are highly active, so that if the amounts present in the quantity of foods normally eaten at one time were injected intravenously, disastrous consequences might be expected. The most prominent amines are tyramine, dopamine, and norepinephrine (Figure 10.15). Serotonin and histamine are also present in significant quantities in some foods. Most of the amines listed in Table 10.24 do not pose a significant health hazard since they are rapidly metabolized (oxidative deamination) by the enzyme monoamine oxidase (MAO) in the body. However, it was realized in the early 1960s that the pressor agent tyramine could produce very serious effects in individuals medicated with

Vasoactive Pressor Amines in Some Selected Foods Amine, µg/g or µg/mla

Plant substance

Serotonin

Tryptamine

Tyramine

Dopamine

Norepinephrine

Banana peel Banana pulp Plantain pulp Tomato Red plum Red blue plum Blue plum Avocado Potato Spinach Grape Orange Eggplant Pineapple juice Pineapple, ripe Pineapple, green Passion fruit Pawpaw

50–100 28 45 12 10 8 0 10 0 0 0 0 0 25–35 20 50–60 1–4 1–2

0 0 — 4 0–2 2 5 0 0 0 0 0.1 0 — — — — —

65 7 — 4 6 — — 23 1 1 0 10 — — — — — —

700 8 — 0 0 — — 4–5 0 0 0 0 — — — — — —

122 2 — 0 + — — 0 0.1–0.2 0 0 + — — — — — —

a

—, food was not tested for this amine; 0, level of the amine was below the detection threshold; +, material contained a trace of the amine. Source: Compiled from Bruce (1961), Foy and Parratt (1960, 1961), Hodge et al. (1964), and Udenfriend et al. (1959).


NH2

NH2 HO

Phenyl ethylamine

Tyramine OH HO

HO

NH2

NH2

HO

HO

Dopamine

Norepinephrine OH

OH HO NH2

NH HO

HO

Octopamine

H3C

Epinephrine

OH

NH HO

CH2CH2N(CH3)2

H 3C

Synephrine Figure 10.15

HO

Hordenine

Chemical structures of some important vasoactive pressor amines commonly found in foodstuffs.

drugs that inhibit MAO (Kuhn and Lovenberg, 1982). MAO inhibitors (MAOIs) are often prescribed for people with depressive illnesses. Ingestion of foods containing large amounts of tyramine by individuals medicated with an MAOI can lead to sporadic bouts of hypertension, intense headache, and, in severe cases, intracerebral hemorrhage and death (Blackwell et al., 1967). Because of MAO inhibition, the ingested tyramine is not readily metabolized; nor is norepinephrine, which tyramine can release. Fortunately, the prevalence of hypertensive crises for individuals medicated with MAOIs after eating foods containing pressor amines is quite low, reportedly 8.4% (Bethune et al., 1964).


Another vasoactive amine causing severe headaches is phenyl ethylamine, which is present in chocolates, cheeses, and red wines (Sandler et al., 1974). As little as 3 mg of phenyl ethylamine has been shown to provoke migraine headache in 50% of subjects compared to 15% after placebo challenge. The mechanism by which these amines cause headache is unknown. Both amines are present in human brain and may function as modulators of catecholamine formation (Concon, 1988). Tyramine causes a release of norepinephrine from its binding sites, and its hypertensive effects may be related to this process. Phenyl ethylamine causes dramatic changes in blood flow through the brain.

Other amines not listed in Table 10.24 include synephrine and octopamine, both found in lemons and bananas, and hordenine, found in barley, particularly on germination. All these compounds are hypertensive agents and psychostimulants and in large doses cause convulsions and respiratory failure (Concon et al., 1979). Vasoactive amines can also be formed by metabolic transformation of precursors that are endogenously present in plant foodstuffs. Thus, dihydroxyphenylalanine (DOPA) is present in broad beans (Vicia faba) (Hodge et al., 1964). This compound has been implicated in hypertensive episodes that followed the consumption of broad beans by patients on MAOIs. DOPA appears to have been found only in the pods and seeds of certain legumes. It is decarboxylated to dopamine, which is responsible for the pressor activity.

10.19 PSYCHOACTIVE SUBSTANCES The psychoactive agents in foods are generally divided into nonnitrogenous and nitrogenous compounds. The first category includes phenylpropenes, such as myristicin from Myristica fragrans, a tree that yields the spices nutmeg and mace. After the ingestion of 5–15 g (several teaspoons) of powdered nutmeg, many people report acute confusional states with visual hallucinations and distortions of time and space. The psychoactive episode produced by nutmeg is frequently followed by abdominal pain and, in some cases, depression and stupor. Poisoning and death due to fatty degeneration of liver have also been documented after nutmeg ingestion (Kreig, 1964). Other substances that probably also contribute to the psychoactive effects of nutmeg include eugenol, geraniol, safrole, borneol, and elemicine (Kuhn and Lovenberg, 1982). The nitrogenous group of psychoactive substances includes phenyl ethylamines, tropanes, tryptamines, and xanthines. The chemical structures of some of the important toxicants in this class are shown in Figure 10.16. Mescaline (3,4,5-trimethoxyphenethylamine) is found in peyote cactus or peyotl, the dried crown of which is often eaten in parts of Mexico. Dioscorine is a tropane alkaloid found in several species of yam. It is a central nervous system depressant and convulsant (Concon, 1988). In humans, dioscorine produces a burning sensation in the mouth and throat, abdominal pain, vomiting, diarrhea, and speech disturbances. These symptoms are followed by vertigo, salivation, lachrymation, sensation of heat, exosphthalmos, deafness, and delirium. In severe cases, death may occur (Corkill, 1948). Yams of Dioscorea du-


metorum and D. hispida have been implicated in many cases of poisoning. Another psychoactive compound found in carrots and celery is carotatoxin. This acetylenic alcohol (Figure 10.16) resembles the powerful central nervous system (CNS) stimulant cicutoxin, a very poisonous substance from the water hemlock (Cicuta masculata) (Anet et al., 1953). Carotatoxin is highly neurotoxic to mice. The hallucinogenic tryptamines are pharmacologically a potent class of compounds (Figure 10.17) and are found in a variety of mushrooms, the most famous of which is perhaps Psilocybe mexicana. Several case reports from Great Britain in the clinical literature have described anxiety and panic attacks as well as schizophrenia-like symptoms after ingestion of psilocybin mushroom (Hyde et al., 1978; Benjamin, 1979). The hallucinogenicity of these mushrooms has been attributed primarily to psilocybin (N,N-dimethyl-4-hydroxytryptamine-O-phosphate) and psilocin (N,N-dimethyl-4-hydroxytryptamine). Another mushroom, fly agaric (Amanita muscaria), contains bufotenine (N,N-dimethyl-5-hydroxytryptamine), which is also a potent hallucinogenic agent.

CH2CH2NH2 OCH3

Mescaline H3CO OCH3

O O

N CH3

(C9H17)C

Dioscorine CH3

C C C CH2

CH CH

CH2

OH

Carotatoxin Figure 10.16

Some psychoactive compounds found in foods.

HO NH2

NH2

N H

N H

Tryptamine

Serotonin HO NH

N H

N N H

H 3C

N-Methyl tryptamine

H3C

Bufotenin

OH

O

PO3H2

N N H

H3C

CH3

N CH3

H3C

N H

Psilocin

CH3

Psilocybin H3CO N

N N H

H3C

CH3

Dimethyltryptamine Figure 10.17

N H

H3C

CH3

5-MethoxyN,N-dimethyltryptamine

Chemical structures of tryptamine and its derivatives.

10.20 METHYLXANTHINES The most common methylxanthines found in our food supply are caffeine (1,3,7-trimethylxanthine) and theobromine (3,7-dimethylxanthine). Theophylline (1,3dimethylxanthine) is only a minor dietary constituent, although it is ingested commonly as a therapeutic agent. These compounds are all methylated derivatives of xanthine, a dioxypurine, and differ only in the number and placement of their methyl groups (Figure 10.18). Caffeine is the most prevalent methylxanthine in our diet. It is found in as many as 60 plant species throughout


the world. Historically, caffeine from natural sources has been consumed and enjoyed from very early times; tea is the oldest recorded caffeine-containing beverage. Perhaps the universal consumption of beverages that contain these substances indicates that they are of no significance. Nonetheless, these are toxicants and their effects are not generally appreciated. Caffeine content of various beverages is summarized in Table 10.25. On the basis of the beverage consumption data from the Market Research Corporation of America (MRCA), the mean caffeine intake from coffee for adult users ranged from 2.74 to 3.98 mg/kg body weight per day (Barone and Roberts, 1996).

O H3C

Table 10.25

CH3 N

N

Caffeine O

N

O

O

H N

N

Theophylline N

Beverage or food Prepared coffee (drip, vacuum, percolated) Instant coffee Decaffeinated coffee, instant Decaffeinated coffee, ground Tea Cola drinks Cocoa

N

CH3

H3C

Caffeine Content of Various Beverages Caffeine content, mg/cup 90–500 60–100 1–4 2.6 60–75 40–60 0

Source: Compiled from Concon (1988) and Emerson and Chappel (1999).

N

CH3

O H

CH3 N

N

Theobromine O

N

N

CH3

Figure 10.18 Chemical structures of methylxanthines commonly found in foods.

For tea, the comparable caffeine intake range was 0.9–1.4 mg/kg body weight per day and for colas, 0.23–0.46 mg/kg/day. The methylxanthine content of beverages varies considerably. Caffeine is the only methylxanthine in coffee; tea contains caffeine, theobromine, and theophylline in decreasing order of concentration, respectively; in cocoa, the predominant methylxanthine is theobromine, with a smaller amount of caffeine and a trace of theophylline. Caffeine is the only methylxanthine added to the soft drinks at concentrations of 15–29 mg/6-oz serving (NSDA, 1982; Emerson and Chappel, 1999). Methylxanthines are vasoactive compounds. Jick and associates (1973) suggested that long-term coffee drinking might be associated with an increased risk of myocardial infraction. Caffeine also has other effects on the cardiovascular system, including decreases in heart rate (Coltan et al., 1968) and increases in blood pressure and cardiac output, probably because of its ability to increase (by inducing the release) the urinary concentration of the pressor amines epinephrine and norepinephrine (Atuk et


al., 1967; Bellet et al., 1969). In a double-blind random cross-over design, Robertson and colleagues (1978) demonstrated that the administration of 250 mg caffeine to noncoffee drinkers increased plasma rennin activity by 57%, and plasma norepinephrine and epinephrine activity were increased by 75% and 207%, respectively. Blood pressure also rose 14/10 mm Hg 1 hour after caffeine ingestion. The acute toxicity of methylxanthines has been thoroughly characterized in several animal species (Table 10.26). The most common identified cause of death is respiratory failure after convulsion. Dogs are more sensitive to the toxic effects of theobromine than other animal species, presumably because of their slower elimination rate. In humans, methylxanthine toxicity manifests itself as a disturbed state of consciousness, followed by convulsions, vomiting, and coma. Death has been reported to follow ingestion of massive doses. The possible mutagenicity and carcinogenicity of coffee, tea, or the related methylxanthines have been extensively reviewed (IARC, 1991; Haynes and Collins, 1984; Aeschbacher, 1991; Mohr et al., 1993). Carcinogenicity studies of coffee, tea, cocoa powder, and caffeine in a variety of animal models under various experimental conditions and dose levels have been reported and are summarized in Tables 10.27 and 10.28. These studies provide convincing evidence that these compounds are not carcinogenic in laboratory animals. Evidence continues to accumulate that behavioral differences exist between caffeine consumers and abstainers. These differences may be attributed to effects of methylxanthines at the adenosine receptor site, since ingestion of caffeine from dietary sources could potentially produce blood caffeine levels high enough to compete with endogenous adenosine at its receptor site.

Table 10.26

Acute Toxicity of Methylxanthines and Their Derivatives LD50, mg/kg

Compound Methylxanthines Caffeine

Theobromine Theophyllinec Methylxanthine dervatives Caffeine Caffeine and sodium benzoate Theobromine Theobromine and sodium acetate 1-Allyl theobromine 1-Butyl theobromine 1-Isoamyl theobromine Theophylline Theophylline ethylenediamine (aminophylline) 7-Allyl theophylline 7-Butyl theophylline 7-Isoamyl theophylline

Species

Oral

i.p.

Clinical signs

Human Rat Mouse Hamster Guinea pig Rabbit Cat Dog Rat Mouse Rat Mouse

150–200a 200 127 230 230 246 125b 145b 950 1350b — 332

— 200 220b — 235 — 190b — — 789 206 217

Convulsions, emesis, coma Convulsions, respiratory failure Convulsions Convulsions Convulsions, stupor Convulsions Convulsions Convulsions — — Delayed convulsions, titanic spasm —

Mouse Mouse Mouse Mouse Mouse Mouse Mouse Mouse Mouse

127 878 950 1356 191 667 772 332 391

220b 525 — 789 102 230 222 217 257

Convulsions

Convulsions Emesis Convulsions Profuse salivation, emesis, convulsions

Mouse Mouse Mouse

315 617 723

299 272 211

Convulsions Convulsions, emesis

Emesis

a

Fatal dose. Median lethal dose. c Theobromine sodium acetate was used because of poor solubility of theobromine. Source: Compiled from Tarka (1982) and Tarka and Shiveley (1987). b

There is also a long history of concern about the safety of beverages containing caffeine. In 1958, caffeine was placed on the U.S. Food and Drug Administration (US FDA) Generally Recognized as Safe (GRAS) list. However, as part of its 1978 review of all GRAS substances, this agency recommended that additional research be done to resolve any uncertainties about the safety of caffeine (FASEB, 1978). In 1980, the FDA proposed that caffeine no longer be considered a GRAS substance, but that it be placed in an interim food additive status pending the completion of additional studies. The areas that raised most concern were the potential reproductive, teratogenic, mutagenic, carcino-


genic, and behavioral effects of the methylxanthine. Several of these aspects of methylxanthine toxicity have been reviewed (Tarka and Shively, 1987; Concon, 1988). At present, the overwhelming evidence available provides ample assurance that sane dietary intakes of methylxanthines have no substantial deleterious effects on health. Reports and allegations of serious bad effects have consistently not been sustained by careful research studies. The task of assessing whether there are slight and inconstant deleterious effects of dietary caffeine on health or performance is difficult because assessment of all slight effects in people is often difficult.

Table 10.27

Carcinogenesis Assays of Coffee, Tea, and Cocoa

Test material Assam leaf tea (tannin fraction) Assam leaf tea (aqueous extract) Instant coffee

Dose

Route

Duration

Animals, no.

8 mg/Wk

Subcutaneous

45–77 Wk

15 M, 15 F

12 mg/Wk

Subcutaneous

69–70 Wk

15 M, 15F

0%, 1%, 2.5%, 5% W/W 0%, 25%, 50%, 100%

Diet

150 M, 150 F

0 and 120 mg/kg/Day 0%–5%

Drinking water Diet

During gestation, lactation,a 720 days post weaning For 5 Wk before mating; during gestation, during lactation, and for 2 yr after weaningc 103 Wk

Instant coffee, regular coffee, decaffeinated coffee, decaffeinated coffee plus caffeine

0%, 6%

Cocoa powder

0%, 1.5%, 3.5%, 5%

Brewed coffee

Brewed coffee Instant coffee solids

a


NIH black rat, 1–2 months NIH black rat, 1–2 months Weanling Swiss mice

Results Local malignant histiocytomas No increase in tumor incidence

55 M 55F

Sprague-Dawley rats, 5–6 weeks

Benign liver adenomasb, growth impaired and survival improved at higher dose levels Tumor incidence significantly increased only at lowest dose level

C 25 M, T 25 M

C57 BL/6J mice

No increase in tumor incidence

2 yr

C 41 M, 41 F T 144 M, 144 F

Sprague-Dawley rats, 21 days

Diet

2 Yr

C 40 M, 40 F T 40 M, 40 F per group

Sprague-Dawley rats, 100 g body weight

Diet

During gestation, lactation, and 104 weeks post weaning

90 M, 90 F (50 M, 50 F additional control group)

Sprague-Dawley rats

Urinary bladders of 94 M and 99 F treated and 29 M and 29 F control examined histologically; no hyperplasia or bladder tumors observed In decaffeinated coffee groups lower body weight and improved survival rate vs. in controls, no increased tumor incidence in any treatment group No evidence of carcinogenicity

Drinking water

Maternal females were fed 1% instant coffee in the diet during gestation and lactation. Two hepatocellular carcinomas were observed in one male mouse fed 1% coffee and in one male mouse fed 2.5% coffee. c Maternal females were fed 50% coffee in the drinking diet. Source: Emerson and Chappel (1999). b

Species and age

Table 10.28

Carcinogenesis Studies of Caffeine

Dose

Route

Duration

Drinking water

No. of animals

Species and age

C 50 M, 50 F T 50 M, 50 F 32 M, 32 F

ICR mice

0%, 0.04%, 0.1%, 0.2% 0 and 100 mg/kg Body weight 0%, 0.1%, 0.2%

Gavage

M 60 weeks F 104 weeks Lifetime

Diet

117 Weeks

C 30 M T 30 M

0%, 0.1%, 0.2%

Drinking water

104 Weeks

C 50 M, 50 F T 50 M, 50 F

Wistar rats

0%, 0.2%

Drinking water

12 Months

C 40 F T 40 F

Wistar rats

0%, 0.02%, 0.43%, 0.093%, 0.2%

Drinking water

104 Weeks

C 50 M, 50 F (two groups) T 50 M, 50 F

Sprague-Dawley rats

0%, 0.25%, 0.5%

Drinking water

43 Weeks

37–43 F

C3H mice

Sprague-Dawley rats Sprague-Dawley rats

Results Tumor incidence not significantly affected by caffeine No effect on tumor incidence of caffeine Tumor incidence not significantly different in treated rats and controls Tumor incidence for any organ site not different in treated animals and controls Number of pituitary adenomas significantly greater in treated rats than in controls No significant increase in tumor-bearing rats or tumors of specific sites in treated rats vs. controls Incidence of mammary tumors, time to tumor appearance not affected by caffeine; number of mammary tumors in affected mice increased significantly at 0.05% dose level

Source: Emerson and Chappel (1999).

10.21 PYRROLIZIDINE ALKALOIDS The pyrrolizidine alkaloids form a group of some 200 different compounds. Plants containing pyrrolidizines have been responsible for numerous outbreaks of poisoning of livestock causing serious economic damage. In recent years, they have been identified as causing human deaths in less developed countries through contamination of cereal crops and harvested seed. They also have been suspected of causing illness after intentional ingestion as vegetables and in the form of herbal remedies. Pyrrolizidines are found mainly in the families of Compositae, Boraginaceae, and Leguminosae, but also in Apocyanacae, Ranunculaceae, and Scrophulariaceae. Some species may essentially contain only a single pyrrolizidine alkaloid but most contain between five and eight. The pyrrolizidine structure is based on two fused five-membered rings that share a bridgehead nitrogen atom, forming a tertiary alkaloid. In nature the rings are most frequently substituted with a hydroxymethylene


group at position C-1 and a simple hydroxyl group at position C-7, forming a structure known as necine base. The bases most commonly encountered are heliotridine and retronecine, which differ only in their configuration about C-7. The chemical structures of several important representatives are also shown in Figure 10.19. The two most significant sources of exposure of humans to pyrrolizidine alkaloids are the accidental contamination of foodstuffs and the intentional ingestion of plants containing pyrrolizidine alkaloids in the form of culinary vegetables or herbal medicines. Serious and wide-scale incidents of illness have been reported through contamination of cereal grains with the seeds of pyrrolizidine alkaloid-containing plants (Crews, 1998). Similarly, many plants that contain these alkaloids are deliberately consumed as food or herbal remedies in all parts of the world. For example, in Japan, plants of Petasites, Symphytum, and Tussilago spp. are eaten as vegetables, although in other countries the major uses are medicinal. As a result of increasing interest in “alternative” therapies and herbal medicines in Europe and the United States, preparations of

A

B

CH2OH

HO

O

O

O

CH3

O

CH3

O

H 3C

O

H3C

CH2OH

HO

O

O

CH2

CH2

N N O

C

H3C

D

HO

H3C

CH3 HO

O

O

O

O

CH3

CH3

HO

CH3

O O

O

H3C

CH2

HO

CH3

O

OCH3 N

N

E

CH3

O

H

F

O

H3C

H O

OH CH3

O

O

HO

CH3

H3C

O

O

CH3 O

CH2

O

O

N

N

CH3

G

H3C

H HO

CH2OH

N

CH3CO

O

CH2OCO

CH3

C

CH

OH

OH

CH3

N

Figure 10.19 Structures of carcinogenic pyrrolizidine alkaloids: A, retrorsine; B, isatidine; C, monocrotaline; D, lasiocarpine; E, petasitenine; F, senecionine; G, dehydroretronecine; H, acetyllycopsamine.


pyrrolizidine alkaloid–containing plants have become widely available commercially and have been publicized for their health-giving properties. Comfrey (Symphytum officinale) in particular has long been a popular herb. Consumers of comfrey could be ingesting up to 5 mg of these alkaloids per day (Speijers and Egmond, 1999). The toxicity and metabolism of pyrrolizidine alkaloids have been extensively studied and reviewed (Winter and Segall, 1989; Segall et al., 1991; Cheeke and Huan, 1995; Crews, 1998). Several have been shown to be hepatotoxic and hepatocarcinogenic in animal studies, and they represent a potential cause of cancer in humans (WHO, 1988). Consumption of herbs containing pyrrolizidine alkaloids might be associated with the high incidence of chronic human liver disease, including primary liver cancer, in Asian and African countries, especially as they may act synergistically with the hepatotoxic agents aflatoxin and hepatitis B virus (Arseculeratne et al., 1981). Some toxicity data are available, such as data on the activation of the liver, vascular lesion in the lung accompanied by pulmonary hypertension, chronic liver diseases in animal experiments, and acute venoocclusive effects and liver cirrhosis in humans; neurologic effects have also been reported. Children seem to be particularly vulnerable. However, further studies on the exposure and toxicity of many pyrrolizidine alkaloids are required to make a meaningful risk assessment possible. The risk of large-scale poisoning through cereal contamination, however, remains serious in view of the continuing practice of consuming poor-quality grain in times of drought and famine.

10.22 PHYTOESTROGENS Estrogens are steroidal compounds produced in the mammalian body that serve to maintain female sexual characteristics; of them the main human estrogen is 17β-estradiol (Figure 10.20). Phytoestgrogens are a group of nonsteroidal compounds produced by a range of plants that, although lacking the classic steroid ring structure (Figure 10.20), have properties similar to those of 17β-estradiol. In some plant-derived foods, phytoestrogen concentrations are high, and thus their mode of action and possible implications to human health are of interest. Estrogenic activity has been reported in grasses and feedstuffs as well as in fruits, vegetables, cereals, and oils. Well-known examples include apples, cherries, carrots, garlic, parsley, potatoes, barley, corn, oats, rice, wheat, and oils from soy, coconut, peanuts, and olives. Phytoestrogen levels of selected foods are summarized in Table 10.29. The compounds that are most likely to be responsible for the estrogenic activity of plants and plant products


Steroid skeleton

OH

17β-Estradiol

HO

Figure 10.20 Classic steroid ring structure of estrogen and the main human estrogen, 17β-estradiol.

Table 10.29 Foods

Phytoestrogen Levels (µg/g Sample) in Selected

Food Soybean seeds, dry Soy flour Black soybean seeds, dry Tofu Soybean seeds, fresh, raw Green split peas, dry Clover sprouts Kala chana seeds, dry Soybean hulls Cowpea seeds, dry Small white bean seeds, dry Garbanzo seeds, dry Pink bean seeds, dry Small lima bean seeds, dry Yellow split peas, dry Mung bean seeds, dry Great Northern bean seeds, dry Pinto bean seeds, dry Red bean seeds, dry Green beans, fresh, raw Round split peas, dry Alfalfa sprouts Large lima bean seeds, dry a

Isoflavonesa

Coumestrol

1953.0 1777.3 1310.7 278.8 181.7 72.6 30.7 19.0 18.4 17.3 15.6 15.2 10.5 9.2 8.6 6.1 6.0 5.6 3.1 1.5 ND Trace Trace

NDb ND ND ND ND ND 280.6 61.3 ND ND ND ND ND ND ND ND ND 36.1 Trace ND 81.1 46.8 14.8

Isoflavones: daidzein + genistein + formononetin + biochanin A. ND, not detected. Source: Compiled from Concon (1988) and Rickard and Thompson (1997b).

b

are the isoflavones, the coumestans, and the resorcyclic acid lactones (Figure 10.21). Genistein, genistin, daidzen, biochanin A, formononetin, and pratensein are the isoflavones most common in food plants. The two most important coumestans are coumestrol and 4′-O-methyl coumestrol; zearalenone and zearalenol are the two resorcyclic acid lactones most likely to occur in plant products. The latter two are produced by various species of Fusarium molds found in food and fodder plants (de Nijs et al., 1996). Thus, zearalenone and its metabolites may also find their way into the human food chain. The biological potencies of various estrogens have been compared in their affinity for estrogen receptors. The comparative binding affinity for receptors in rat uterine cytosol is 17β-estradiol > coumestrol > zearalenone > genistein > daidzein > biochanin A > formononetin (Verdeal et al., 1980; Aldridge and Tahourdin, 1998). Competition for binding sites in steroid-binding globulin is 17βestradiol > genistein > formononetin > coumestrol > zearalenone (Martin et al., 1978). The physiological effects of estrogens include hypertrophy of the vagina, uterus, and mammary glands in female mammals and hypertrophy of the accessory glands and development of female secondary characteristics in male mammals. The degree to which these signs develop

Isoflavones

depends on the amount of estrogen present, the duration of exposure to them, and the species of the animal. All these effects are usually temporary and disappear with a change to a diet free of estrogens, unless the animals have been exposed to a high level of these compounds for a prolonged period. Estrogens are also implicated in the induction of cancer; the carcinogenic risk is related to the extent to which estrogens are present in food as well as their inherent biological potency (Stob, 1983; Quattrucci, 1987; Aldridge and Tahourdin, 1998). However, since most of the naturally occurring estrogenic substances show only weak activity, it is doubtful that the normal consumption of foods known to contain estrogens would provide sufficient amounts of these substances to elicit a physiological response. Beneficial effects have also been ascribed to the consumption of phytoestrogens, largely on the basis of observed differences in chronic disease between Asian and Western populations. Apart from the beneficial effect on plasma cholesterol concentration and possibly coronary heart disease, other benefits appear less well documented. Soy-containing foods also seem to have beneficial physiological effects in postmenopausal women. Further research on the beneficial effects of phytoestrogens in selected population segments is thus definitely warranted.

Coumestans

R4 R3 R1

R2

O

HO

O

O

O

O

OR

R1

R2

R3

R4

Coumestrol

Daidzein

H

OH

OH

H

4-Methoxycoumestrol

Daidzin

H

O-Glu

OH

H

Genistein

OH

OH

OH

H

Genistin

OH

O-Glu

OH

H

Formononetin

H

OH

OCH3

H

Biochanin A

OH

OH

OCH3

H

Pratensein

OH

OH

OCH3

OH

Prunetin

OH

OCH3

OH

H

R=H

Resorcyclic Lactones OH

O

H

CH3

O HO

O Zearalenone

Figure 10.21

Structure of some typical plant estrogens.


R = CH3

10.23 ALLERGENS Food allergy and food intolerance are terms that are frequently used interchangeably, but they are not the same reaction. Food intolerance refers to any kind of reproducible, unpleasant reaction to a specific food (Angus, 1998). A subgroup of food intolerance is food allergy. A reaction can only be called allergic if it is caused by an immunological response, often involving immunoglobulin E (IgE) antibodies that bind to the food. Allergy is thought to account for less than 20% of all adverse food reactions. Allergens are common constituents of foods and, in the case of soy protein and some tree nuts, are used increasingly in the diet. In contrast to antinutritional and toxic factors associated with foodstuffs, allergens display their effects only in those individuals who have an altered reactivity (allergy) to otherwise innocuous substances. Allergy symptoms usually include flushing of the face, skin disorders, respiratory problems, and gastrointestinal disturbances. The intensity of reaction depends on the degree of hypersensitivity of the individual consuming the allergen rather than the quantity consumed (Perlman 1980; Taylor et al., 1989; Angus, 1998). In true food allergies, the known allergens are usually naturally occurring proteins, glycoproteins, or polypeptides found in the food (Taylor et al., 1987, 1989). The allergenicity can be associated with the type of structure of the proteins and the peptides as primary, secondary, or tertiary. In the case of tertiary structures, allergenicity often disappears on denuatration, whereas it is retained in the case of primary structures. Furthermore, the protein has to be large enough to be recognized by the immune system as a foreign compound. As a general rule, the allergenicity of molecules with a molecular mass lower than 5000 is low, unless they are bound to endogenous proteins. On the other hand, substances with a molecular mass higher than 70,000 are not absorbed and do not have contact with the immune system. Some common allergenic foods are listed in Table 10.30. A few food allergens have been purified and characterized (Table 10.31). Soy and other vegetable and tree nut proteins are among the most common causes of hypersensitivity reactions. Peanut is named as the vegetable protein with the greatest allergenic potential among legumes (Perlman, 1980). Furthermore, sensitization to one legume plant protein may sometimes cause sensitization to another plant of legume origin (Brandon et al., 1986). The increased use of soy protein products in prepared foods and feeds has raised concern about the possible immunological consequences of a high intake of soy protein in humans (Heppell et al., 1987).


Table 10.30

Common Allergenic Foods

Infants Cow’s milk Eggs Legumes (peanuts and soybeans) Wheat Adults Legumes (peanuts and soybeans) Crustaceans (shrimp, crab, lobster) Molluscs (clams, oysters, scallops) Fish Tree nuts (almonds, Brazil nuts, cashews, hazelnuts, macadamia nuts, pecans, pine nuts, pistachios, walnuts) Eggs Wheat Miscellaneous (coconut, sunflower seed, nut oils)

Only a few studies detail the size of the soy allergy problem, its incidence and prevalence, the mechanism of the allergy reaction, and the factors responsible. Immunoassays have been used to identify the soy protein fractions responsible for the allergic reactions in humans. The two storage globulins of soybeans, glycine (11S) and βconglycinin (7S) and their subunits, as well as proteins from the 2S fractions, such as the Kunitz inhibitor, are involved in the allergic reactions in humans (Table 10.31). It is yet unclear whether soy oil can provoke allergic reactions in soy-sensitive individuals. Processing conditions can often influence the concentration of antigens and aller-

Table 10.31

Allergenic Food Proteins

Cow’s milk: casein, β-lactoglobulin, α-lactalbumin, others Egg whites: ovomucoid, ovalbumin, ovotransferrin (conalbumin) Egg yolks: lipoprotein, livetin Peanuts: peanut I, lectin-reactive glycoprotein, arachin, conarachin Soybeans: β-conglycinin (7S fraction), glycinin (11S fraction), 2S fraction, Kunitz trypsin inhibitor, unidentified 20-kD protein Codfish: allergen M (parvalbumin) Shrimp: antigen II Green peas: albumin fraction Rice: glutelin fraction, globulin fraction Cottonseed: glycoprotein fraction Tomato: several glycoproteins Source: Compiled from Taylor et al. (1989) and Deshpande and Sathe (1991).

gens by altering the immunochemical structure of proteins and by influencing the digestibility of antigenic proteins (Pedersen, 1986). The epitopes of soy agents may be affected in a variable manner according to the conditions encountered during food processing. The antigenic activities of both glycinin and β-conglycinin can be eliminated by physicochemical denaturation of these proteins (Sissons et al., 1982). The literature on tree nut allergens has been reviewed by Angus (1998). Allergenic responses are seen in sensitive individuals who consume almonds, Brazil nuts, cashews, hazelnuts, macadamia nuts, pecans, pine nuts, pistachios, walnuts, coconut, and sunflower seeds. In many cases, the allergens are found to be IgE-binding proteins. In the case of food allergy, late reactions seldom occur. The clinical symptoms of allergic food reactions are listed in Table 10.32. The oropharynx and gastrointestinal tract are the initial sites of exposure to food antigens. Symptoms such as edema and itching of the mouth often occur. However, these reactions may be transient and are not necessarily followed by other symptoms. In some individuals, certain fruits, nuts, and vegetables cause oral symptoms only; in others a more extensive reaction is seen. The quantity of the offending food also plays a role in the gravity and extent of reaction, although in principle a small amount of a certain food can readily cause a response. Sometimes the allergic reaction develops only if the food intake is followed by exercise. This is referred to as exercise-induced food allergy. Hypotension and shock are life-threatening consequences of a food-allergic reaction. Generally, the reaction is accompanied by other anaphylactic symptoms such as abdominal cramps, nausea, vomiting, diarrhea, dyspnea, urticaria, and angioedema.

Table 10.32

Symptoms of Food Allergy

Skin symptoms Itching, erythema, angioedema, urticaria, increase of eczema Respiratory symptoms Itching of eyes, nose, throat; tearing and redness of the eyes; sneezing; nasal obstruction; swelling of throat, shortness of breath; cough Gastrointestinal symptoms Nausea, vomiting, abdominal cramps, diarrhea Systemic symptoms Hypotension, shock Controversial symptoms Arthritis migraine, glue ear, irritable bowel syndrome


Similar to food allergy, food intolerance comprises many different clinical disease entities, with different symptoms. Often, the clinical picture is difficult to distinguish from that of an allergic reaction. The distinction intolerance/allergic, therefore, cannot always be made on the basis of the individual’s history alone. The manifestation of food allergy and food intolerance can vary from innocent symptoms, such as rhinorrhea, to life-threatening symptoms, such as shock. The diagnosis is made on the basis of clinical as well as laboratory data, according to the following general procedure: 1.

2.

3.

4. 5. 6. 7. 8.

History of the patient (complaints, possible associations with food intake, family history, atopic manifestations) Overview of food intake, recorded by a dietician (often, people have already excluded food products of their own accord) Physical examination (signs of eczema, asthma, rhinitis, abdominal disorders, and nutritional state) Blood examination (food allergens, inhalant allergens) Skin tests Exclusion of all potentially suspected foods (trial diet) Challenge test, for one or a few food products or additives Gradual reintroduction of food products

Some of the diagnostic tests are rather time-consuming and costly. In addition, they cause some risk or discomfort to the patient. The approach is modified according to the type of reaction involved, age, and other characteristics of the patient. Skin test results and specific IgE determinations may be unreliable. For many foods, the identity of the allergenic moieties is unknown and information about their stability is lacking. Examples include the allergens of some fruits and vegetables. Negative results do not rule out a possible allergy for the tested allergen, and, positive results do not automatically imply that the particular food does indeed cause the symptoms. The gold standard for the diagnosis of food allergy remains reintroduction or challenge after a period of exclusion. If the diagnosis is correct and compliance is maintained, exclusion of the suspicious food(s) shuld result in improvement in the patient’s condition, and challenge or reintroduction should lead to relapse of symptoms. Food intolerance, in contrast, is not IgE-mediated and cannot be detected by skin tests and specific IgE determinations. It can only be demonstrated by exclusion and challenge. The preferred approach to the management of food allergy is

prevention. Once the treatment is started, strict avoidance of all offending foods is needed. In sensitive individuals allergic responses to use of these ingredients in food products have created special problems in the food industry. The industry is becoming increasingly aware of the special attention required when manufacturing nut-containing and other foods in the same factory. A large number of food manufacturers have altered their production processes to reduce the risk of crosscontamination. Warnings on food labels are also being increasingly used to alert customers to the presence or absence of nuts in prepared food products.

10.24 ANTIVITAMINS A number of substances have been reported to act on the availability of vitamins and are commonly referred to as antivitamins. An antivitamin can be defined as any compound that under certain conditions can actually or potentially produce toxic symptoms such as in a deficiency of a corresponding vitamin, whether administered parenterally, ingested, or occurring along with the vitamin in food (Concon, 1988). According to this definition, a substance that degrades a vitamin is also considered an antivitamin even though deficiency symptoms have not been demonstrated because of its presence in the diet. These substances may possess only the potential to produce deficiency symptoms, especially when present in large amounts, and must be examined when vitamin intakes from food sources are considered. The definition also includes nutrients that when taken in excess increase the physiological requirement of other vitamins. The deleterious effects of antivitamins may be due to one or several of the following factors: They may have similar chemical structures, they compete with the vitamin in various metabolic reactions, they may react with the vitamin per se and make it unavailable, or they may give rise to symptoms resembling vitamin deficiencies. Somogyi (1978) has proposed dividing the antivitamins into two major groups: (a) structurally similar compounds that compete with the vitamins as a result of the resemblance of their chemical structure and (b) structuremodifying chemicals that destroy or decrease the effects of a vitamin by modifying the molecule or by forming a complex. Most antivitamins occurring in the natural food chain belong to the latter group. The first antivitamin to be described seems to be thiaminase, which was reported in the mid-1930s (Green, 1937). Antithiamine factors are found in many fish species and in certain species of crab and clam. A thiamine inactivity effect has been described


in some fruits and vegetables, such as blueberries, black currants, red beets, Brussels sprouts, and red cabbage. The thiaminase enzyme splits thiamine at the methylene linkage (Figure 10.22). The enzyme contains a nonprotein coenzyme, structurally related to hemin, the red pigment component of hemoglobin. The coenzyme is the actual antithiamine factor. Cooking destroys thiaminases in fish and other sources. Antithiamine factors can also be of plant origin. A study of the effects of tannins in tea leaves, tea infusions, and betel nuts on thiamine in volunteers showed that they were responsible for thiamine destruction (Janssen, 1997). The interaction of these substances with thiamine is oxygen-, temperature-, and pH-dependent. Other antithiamine factors include ortho-catechol derivatives. A well-known example is present in bracken. The so-called fern poisoning in cattle is attributed to this factor. Possibly there are two types of heat-stable antithiamine factors in this fern, one of which is identified as caffeic acid (3,4-dihydroxycinnamic acid) (Figure 10.23). Caffeic acid can also be formed on hydrolysis of chlorogenic acid by intestinal microflora. Chlorogenic acid is found in green coffee beans, green apples, and sunflower seeds. Other ortho-catechols, such as methylsinapate (Figure 10.23), which occurs in mustard or rapeseed, also have antithiamine activity. Another example of an antivitamin is ascorbic acid oxidase. This copper-containing enzyme mediates the oxidation of free ascorbic acid (vitamin C) to dehydroascor-

H 3C

Thiamine

CH2

N H3C

CH2CH2OH

NH2 N S N

Thiaminase

NH2 N H3C

H3C

CH2CH2OH

CH2OH N N

S

Figure 10.22 Degradation of thiamine (vitamin B1) by thiaminase.

HO

CH

Methylsinapate

CHCOOH

HO

Caffeic acid

H3CO

CH

CH

COOCH3

HO OCH3

HO

COOH

O HO

CH

CHCO

HO

Figure 10.23

OH OH

Chlorogenic acid

Antiathiamine factors of plant origin.

bic acid and then to diketogulonic acid, oxalic acid, and other oxidation products. The enzyme is present naturally in many fruits and vegetables, including cucumbers, pumpkins, lettuce, cress, peaches, bananas, tomatoes, potatoes, carrots, and green beans. Its activity varies with the type of fruit or vegetable. As an enzyme, ascorbic acid oxidase can be inhibited effectively by blanching of fruits and vegetables. A variety of plants and mushrooms contain pyridoxine (vitamin B6) antagonists; most are hydrazine derivatives. A pyridoxine antagonist, linatine (γ-glutamyl-1amino-D-proline), was identified in linseed meals. This water-soluble, heat-labile compound readily hydrolyzes to the hydrazine derivative 1-aminoproline, the actual antipyridoxine factor (Figure 10.24). Antipyridoxine factors have also been found in wild mushrooms, the common commercial edible mushroom, and the Japanese mushroom shitake. Commercial and shitake mushrooms contain agaritine. It is hydrolyzed in the mushroom by γ-glutamyl-transferase to the active agent 4-hydroxymthylphenylhydrazine (Figure 10.25). The hydrolysis of agaritine is accelerated if the cells of the mushrooms are disrupted. The mechanism underlying the antipyridoxine activity is believed to be condensation of the hydrazines with the carbonyl compounds pyridoxal and pyridoxal phosphate, the active form of the vitamin, which results in the formation of inactive hydrazones.


Other examples of antivitamins include a pantothenic acid inhibitor purified from peas (Kratzer et al., 1954; Smashevskii, 1966). Perhaps the best-known example in this category is avidin, a protein that occurs in raw egg white, which acts as a biotin antagonist.

Linatine HOOC

N

γ-Glutamyl

NH

H2O

1-Amino-D-proline Glutamic acid HOOC

N NH2

Figure 10.24 Hydrolysis of linatine, an antipyridoxine factor found in linseed to the active factor, 1-amino-D-proline.

Agaritine HOH2C

NHNH

CO

CH2

CH2

CH

COOH

NH2

γ-Glutamyl transferase

HOH2C

NHNH3

HOOC

CH2

CH2

CH

COOH

NH2

4-(Hydroxymethyl)phenylhydrazine

Glutamic acid

Figure 10.25 Hydrolysis of agaritine, an antipyridoxine factor found in common edible mushrooms, to the active agent. 4-hydroxymethylphenylhydrazine.

10.25 MISCELLANEOUS ENDOGENOUS TOXICANTS A number of toxicants, which do not fall into any of the categories described, are also found in some specific foods. Some of these are briefly described in the following sections. 10.25.1

D-Mannoheptulose

Avocados are the only known major source of the compound D-mannoheptulose (Figure 10.26). In both human and animal subjects, consumption of avocados has been shown to result in hyperglycemia or a depression in plasma insulin level and the insulogenic index. Coore (1963) has shown that mannoheptulose completely but reversibly suppresses the stimulating effect of D-glucose on insulin secretion. This sugar is potent enough to inhibit the rat pancreas from secreting insulin at doses that do not induce hyperglycemia. 10.25.2 Umbellulone Umbellulone (Figure 10.26) is found in California bay laurel (Umbellularia californica), whose oil contains from 40% to 60% of this compound. It possesses an atropinelike effect on the nerves and muscle fibers of frog heart. Contact with the oil, or even exposure to the vapors, can cause


skin irritation and headache, and in some cases the effect can be serious enough to cause unconsciousness (Concon, 1988). California bay laurel should not be mistaken for the conventional bay leaf (Laurus nobilis), which is devoid of this toxic effect. 10.25.3 Glycyrrhizin Licorice, widely used in candies and other confections as a flavoring agent, contains a glycoside, which constitutes about 5% to 10% of the root. The glycoside consists of two glucuronic acid moieties and a steroidal aglycone glycyrrhetinic acid. The calcium or potassium salt of this glycoside is called glycyrrhizin (Figure 10.26). Because of its steroidal character, the compound possesses marked physiological activity. Licorice has been reported to have a deoxycorticosterone activity, as shown by the usual water and sodium retention and potassium loss that follow consumption (Conn et al., 1968). Excessive consumption of licorice candy can also cause severe hypertension, hypokalemia, aldosteronopenia, suppressed plasma rennin activity, and cardiac enlargement (Conn et al., 1968; Koster and David, 1968). 10.25.4 Menthol Peppermint oil contains approximately 40% menthol. This terpene is widely used as a flavoring agent in candies,

B

CH2OH

A

O

H

CH2OH H3C

OH

OH

CH3 OH

OH H

H3C

H O O H3C

C

C

(C6H8O6)

O

CH3 H 3C CH3

H O CH3

CH3 H

CH3

OH

CH3 O

C

D

(CH2)7

HO H3C

H

NH

CH3

E

O CH3

Figure 10.26

Miscellaneous toxicants found in plants: A, mannoheptulose; B, umbellulone; C, glycyrrhizin; D, menthol; E, Carpaine.

chewing gum, liqueurs, and other products. Besides sensitization reactions in the form of urticaria, excessive consumption of menthol-containing products may cause heart fibrillation and toxic psychosis. Symptoms in such cases disappear when the incriminating products are avoided. 10.25.5 Carpaine Papayas (Carica papaya) contain an alkaloid, carpaine (Figure 10.26). It has a powerful cardiac and diuretic action (Concon, 1988). Carpaine depresses arterial, followed by ventricular contractions of the heart without interference to the conduction system. The depression of cardiac action causes hypotension. At higher concentrations, carpaine causes vasoconstriction, which is not reversed by epinephrine.

10.26 REMOVAL OF TOXICANTS AND ANTINUTRIENTS Traditionally, several methods have been used to remove the toxicants and antinutrients present in plant foods in or-


der to improve their nutritional quality and utilization. To accomplish this goal, several approaches may be considered. Breeding plant varieties containing low or no levels of toxicants is one such approach. Such an approach, however, requires long-term efforts, since the type and number of antinutrients and toxicants present in plant foods are rather large and diverse with respect to their chemical and biochemical nature. Such efforts will also need to consider agronomic consequences of genetic manipulation, including crop yield, soil tolerance, light and water requirements, and resistance to pests and diseases. The physical and chemical means of removing undesirable antinutrients include several processing methods, such as soaking, cooking, germination, fermentation, selective extraction, membrane filtration, irradiation, and enzymatic treatments. In many instances, the use of only one method may not effectively remove all the antinutrients present, and thus a combination of two or more methods may be required to accomplish the desired level of removal. The effectiveness of some of these methods in removing the plant antinutrients is briefly described in the following discussion. Soaking usually forms an integral part of such processing methods as cooking, germination, fermentation,

and roasting. Discarding soak water before further processing can remove several water-soluble antinutrients, such as protease inhibitors, phytates, lathyrogens, goitrogens, cyanogenic glycosides, and polyphenols. The extent of their removal depends upon the soaking temperature, the type of soaking medium, the seed type, the length of soaking, and the solubility characteristics of the soluble antinutrients. Soaking media frequently include water, salt (or combination of salts) solutions, and dilute aqueous alkali solutions. Salt and alkali help leach the solubles into the soaking medium by increasing the cell membrane permeability. However, such loss of antinutrients during soak-

Table 10.33

ing is also associated with loss of desirable nutrients, such as proteins, minerals, and vitamins. Cooking, or heat processing, is probably the oldest known method of processing plant foods for human consumption. Cooking may be done at atmospheric pressure and temperature or at high-pressure and high temperature (autoclaving). The primary purpose of cooking is to render the food palatable and to develop its aroma. The cooking water may or may not be discarded, depending on cultural and personal preferences. Cooking generally inactivates heat-sensitive factors, such as enzyme inhibitors, lectins, volatile compounds such as HCN, and some of the off-fla-

A Summary of the Effects of Antinutrients and Toxicants of Plant Origin

Antinutrient/toxicant

Effects

Proteinase inhibitors Amylase inhibitors

Pancreatic hypertrophy, dietary loss of S-amino acids Amylase inhibition; may hinder carbohydrate utilization Growth depression, fatal

Proper thermal processing Proper thermal processing

Cyanogens

Reduced mineral bioavailability, altered protein solubilition, enzyme inhibition Reduction in protein digestibility and utilization, inhibition of several enzymes Cyanide poisoning; action as progoitrogens

Goitrogens

Inhibition of iodine binding to thyroid gland

Lathyrogens Favism

Neurotoxic, nervous paralysis of lower limbs; fatal Hemolytic anemia

Allergens Saponins Estrogens

Several allergic reactions Bitter taste, foaming, hemolysis Growth inhibition, interference with reproduction

Toxic amino acids

Structural analogs of protein amino acids; act as antimetabolites, potent inhibitors of several enzyme systems Impairment of nervous system; cholinesterase inhibitors; ataxia, convulsions, coma, muscle weakness, fatal Loss of lysine, lowered utilization of dietary proteins, malnutrition, nonspecific interactions with iron and certain enzyme systems

Traditional household processes that remove phytate to a variable degree Traditional household processes that remove polyphenols to a variable degree Proper processing, traditional household methods, breeding for low levels Proper food processing, leaching, breeding for low levels Breeding for low levels, leaching, roasting Breeding for low levels, wet-processing methods, treatment with β-glucosidases Avoidance of foods eliciting allergic responses Leaching with hot water or ethanol Breeding for low levels, reversible effects, avoidance of prolonged consumption of foods containing high levels Breeding for low levels

Phytohemagglutinins (lectins) Phytate Polyphenols (tannins)

Potato glycoalkaloids

Gossypol

Safrole Oxalates

Carcinogenic Renal stones, gastroenteritis, shock, convulsions, low plasma calcium levels, renal damage


Methods of removal

Proper thermal processing

Breeding for low levels

Use of glandless cottonseed varieties, controlled moist heat processing; treatment with organic solvents, aniline and boric acid, chelation using iron Lower dietary intake Leaching, avoidance of prolonged consumption of high-oxalate foods

vor components. The heat-stable factors, such as estrogens, saponins, phytate, polyphenols, and allergens, may not be affected to any significant extent by cooking. Even among the heat-labile antinutrients, complete inactivation may not always be possible. When the soaking and cooking medium (such as water) is not discarded, a significant amount of heat-stable antinutrients and toxicants remain practically unchanged. On the contrary, if the medium is discarded, a significant amount of heat-stable antinutrients can be removed from plant foods. Excessive heat processing, however, should be prevented, since it adversely affects the protein quality of foods. Germination mobilizes reserve nutrients required for the growth of plant seedlings and, therefore, may help in the removal of at least some of the antinutrients, such as phytates and raffinose oligosaccharides (see Chapter 7), which are thought to function as reserve nutrients. Significant reductions in phytate, lectin activity, and raffinose sugars are reported on germination of various legumes (Deshpande et al., 1984a). Beneficial effects of germination in terms of reduction in enzyme inhibitory activities, however, remain controversial. Similarly to those in germination, most of the changes occurring during the fermentation of foods are of a catabolic nature, and they help in the hydrolysis of such components as proteins and carbohydrates. Fermentation of foods can result in significant reduction in the quantity of certain antinutrients. The removal of raffinose oligosaccharides of legume during fermentation, for example, is primarily due to the α-galactosidase activity present in legume seeds as well as in the microorganisms involved in the process. Depending upon the type of legume as well as fermentation, phytic acid is also hydrolyzed during fermentation to a variable degree. In addition to the traditional household processes for preparing plant foods for human consumption, enzymatic methods have been used to remove certain antinutrients of plant origin, including phytates and raffinose sugars. Endogenous enzymes, such as linamarinase, as well as externally added β-glycosidases are often used to remove the cyanogenic glycosides of various legumes. The HCN thus produced is water-soluble and volatile and can be easily removed by heating and/or discarding the soaking water. In addition, processes such as ultrafiltration, irradiation of foods, addition of antibiotics or bacteriostats, extrusion cooking, and protein texturization have proved useful in removing certain toxicants and antinutrients of plant foods. Table 10.33 presents a summary of the commonly occurring antinutrients in plant foods used for human consumption, their antinutritinal effects, and methods of their removal.


10.27 SUMMARY A number of toxic and/or antinutritional factors occur naturally in foods of plant origin. Even those that naturally occur in the animal products usually are derived directly or indirectly from vegetable sources. Food and fodder sources from the plants of the Leguminosae family seem to have the greatest diversity of the toxicants and antinutritional factors. Most of the antinutritional or toxic factors have a pronounced capacity to induce deleterious effects in test animals, when tested by themselves in certain doses. Fortunately, under normal dietary conditions, the concentration of toxicants in the food is too low to cause any adverse effects. The very fact that humans have been using these plants as staple foods over the centuries also suggests that their physiological processes are well suited to handle small amounts of various toxicants in the normal diet. In addition, none of these food plants forms the sole source of nutrients in diets in most parts of the world. We are not likely to have an “ideal food source” that would take care of all our nutritional requirements. The fact that balanced nutrition results from a wise choice of foods of different food groups also minimizes the contribution of any one antinutritional factor from a particular food source. Nevertheless, there is the ever-present danger of adverse circumstances’ forcing major changes in the dietary pattern for a short period. The occurrence of lathyrism in seasons of droughts and floods in certain tribal parts of India is a classic example in this regard. Humans are currently exploring several unconventional food sources to meet the increasing demands and shortages of food supplies. This also means an increased potential risk for the adverse effects of compounds hitherto unknown in the human food chain. Because of the growing concern about the possible direct relationships between diet and disease, we are thus constantly challenged to respond to the general need for exploring the possible adverse consequences of antinutrients and food toxicants and for identifying the factors that contribute to the formation and inactivation of such compounds in our food supply. The basic mechanisms of toxic action of antinutritional factors and toxicants have permitted rational research approaches to develop new ways to minimize their adverse effects. Such approaches include inactivation of deleterious compounds to prevent them from interacting with living cells, proper processing and handling methods to eliminate them altogether from our diet, breeding of new plant varieties that are both nutritious and safe to consume, and identification of dietary constituents that protect against the adverse action of these compounds. The con-

tinuation of our current research efforts in this field can only ensure the certainty of an adequate, wholesome, and balanced diet for all the world’s people.

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11 Fungal Toxins

11.1 INTRODUCTION The toxicity of certain fungi, such as mushrooms, has been recognized for a long time. However, potential human and animal health hazards of other toxigenic fungi were not recognized until the 1850s, when a distinct disease called ergotism was shown to be associated with the ingestion of rye and other cereals infected with the fungus Claviceps purpurea. This was followed by Russian reports of other mycotoxicoses affecting humans. Thus, human stachybotryotoxicosis was linked with the ingestion of bread infected by Fusarium graminearum, and alimentary toxic aleukia (ATA) was associated with the ingestion of overwintered grains infested with Fusarium poae and Fusarium sporotrichioides (Sarkisov, 1954). Although several sporadic cases of mycotoxicoses in domestic animals were reported before 1960, the incidence of turkey X disease in England was the turning point in mycotoxin research and stimulated enormous interest in the study of mycotoxins in the scientific community (Sargeant et al., 1961). More recently, the scientific community was shocked by the discovery of a new group of mycotoxins, the fumonisins, produced by Fusarium moniliforme. Some considered the fumonisins the aflatoxins of the 1990s, because of the dramatic effects of ingestion by horses and pigs and the possibility that they may be involved in human disease (Sydenham et al., 1991; Dutton, 1996). Mycotoxins are secondary fungal metabolites with diverse structures and toxicological properties that induce a variety of toxic effects in humans and animals when foods contaminated with these compounds are ingested.


The toxic effects include acute toxicity, carcinogenicity, mutagenicity, teratogenicity, and estrogenic effects on animals at normal levels of exposure. Biological conversion products of mycotoxins are also referred to as mycotoxins. Although mycotoxins are fungal metabolites, all fungal metabolites are not necessarily mycotoxins. For example, fungi also produce antibiotics, such as penicillin; however, they are not considered mycotoxins. The inherent toxic effects in higher organisms are thus important to the determination of whether a chemical is a mycotoxin. Although many hundreds of such toxic mycotoxins have been identified, only about 20 to 30 have been shown to be contaminants of human or animal food (Watson, 1985). The term mycotoxicosis is applied to pathological conditions that result from the ingestion of foods or feeds contaminated with fungal toxins. Forgacs and Carll, who defined it as “poisoning of the host following entrance into the body of toxic substances of fungal origin,” introduced the term in 1962. During the last four decades, research on mycotoxins has revealed potential hazards of mycotoxin contamination of many important agricultural products. The occurrence of mycotoxins in agricultural commodities depends on such factors as region, season, and conditions under which a particular crop is grown, harvested, and stored. Crops grown in warm and moist weather in the tropical and subtropical countries are much more prone to mycotoxin contamination than those in temperate zones. However, certain toxigenic fungi such as Fusarium species can proliferate at low temperature and produce toxins. Over 100 fungal species have been shown to produce mycotoxins associated with naturally occurring diseases in

animals and humans throughout the world (Rechcigl, 1983; Sharma and Salunkhe, 1991). Although toxigenic fungi and their spores are ubiquitous, mycotoxicosis is primarily a problem in areas that have high rainfall and relative humidity and temperatures that favor fungal growth and mycotoxin production. In addition to specific growth conditions, the fungal spoilage of crops and their grains is enhanced by drought, insect damage, cracking or breaking of kernels during harvesting, and presence of excessive chaff in the harvested grain. During the entire postharvest period, food crops are essentially in a state of storage, and fungal growth on them is preventable only by careful regulation of moisture content, temperature, and other environmental conditions. Mature fruits and vegetables are also highly susceptible to invasion of toxigenic fungi, because they are high in moisture and nutrient content. In addition, many fruits are more easily injured as they approach full maturity and therefore are vulnerable to fungal attack. Postharvest

losses of fresh fruits and vegetables due to microbial decay range from 20% to 50% of total production, depending on the geographic region. Thus fungal infection of foods not only results in economic losses but also is a potential health hazard to humans and animals. Some economically important mycotoxins that contaminate human foods worldwide are listed in Table 11.1. Species of toxigenic fungi can be found in all major taxonomic groups of fungi. Most of the mycotoxins known have been recognized as metabolic products of genera such as Aspergillus, Penicillium, and Fusarium. In addition, other genera, such as Claviceps and other Ascomycetes, are known to produce various mycotoxins. Important toxigenic fungi of several taxonomic groups and their mycotoxins are listed in Tables 11.2 to 11.4. Information on the natural occurrence of mycotoxins in foods and feeds is summarized in Table 11.5. In view of the highly toxic nature of many of the mycotoxins, it is fortunate that they occur in food sporadically and often only in small amounts, at least in the

Table 11.1 Commonly Encountered Toxigenic Fungi, Their Toxins, and Important Agricultural Commodities They Damage Toxin

Fungus

Aflatoxins

Aspergillus flavus A. parasiticus

Ochratoxins

A. ochraceus, Penicillium viridicatum Fusarium spp. (e.g., F. roseum) Fusarium moniliforme Fusarium spp. (e.g., F. tricinctum)

Zearalenone Fumonisins Trichothecenes T-2 toxin Vomitoxin Rubratoxins Citrinin

Tremorgens

Patulin Ergotoxins

P. rubrum P. purpurogenum P. viridicatum P. citrinum A. ochraceus P. cyclopium P. palitans Aspergillus spp. Penicillium spp. Aspergillus spp. Claviceps purpurea C. paspali


Commodities damaged Groundnuts, tree nuts, cereals, cottonseed, soybeans, spices, fruits, feeds such as groundnut meal Legumes, cereals, coffee beans Fungally infected corn, wheat, barley, etc. Corn, other cereals Fungally infected corn, wheat

Fungally infected corn Barley, wheat, rye, oats, rice

Fungally infected feeds, groundnuts, rice

Fungally infected fruits, such as apples, plums, peaches, pears, apricots Fungally infected grains and grasses

Table 11.2 Important Mycotoxins Produced by Aspergillus Species

Table 11.3 Mycotoxins Produced by Penicillium Species

Mycotoxin

Mycotoxin

Producing organism

Citreoviridin Citrinin Penicillic acid Rubratoxins Griseofulvin Luteoskyrin Islanditoxin Mycophenolic acid Decumbin Viridicatin Cyclopiazonic acid Patulin Brevianamide A Penitrems Verruculogen

P. citreoviride P. citrinum P. puberculum P. rubrum P. janczewski P. islandicum P. islandicum P. brevicompactum P. decumbens P. viridicatum P. cyclopium P. patulum P. viridicatum P. cyclopium P. verruculosum

Aflatoxins Sterigmatocystins Ochratoxins Fumigatin Aspergillic acid Kojic acid Terreic acid Helvolic acid Fumagillin Tryptoquivaline Fumitremorgins

Producing organism A. flavus, A. parasiticus A. vesicolor A. ochraceus A. fumigatus A. flavus A. fumigatus A. terreus A. fumigatus A. fumigatus A. clavatus A. fumigatus

developed areas of the world. However, the potential for wide-scale problems exists should the appropriate circumstances arise. Low-level contamination of foods by highly biologically active chemicals presents a major difficulty in assessing their true significance for humans. Legislation is usually set on a national basis and is mainly restricted to the aflatoxins, of which aflatoxin B1 is accepted as a potent liver carcinogen. Some countries also have introduced limits for other selected mycotoxins (FAO, 1997). Various international organizations now pay increased attention to mycotoxins in the human food chain. One development in this regard was the establishment in 1994 of the WHO Collaborating Center for Mycotoxins in Food (WHO-CCMF), located in the Albert Ludwigs University School of Medicine in Freiburg, Germany. A particular task of this center is to elucidate the role of mycotoxin-contaminated food in human health and disease. In 1997, the National Institute of Public Health and the Environment, Bilthoven, The Netherlands, was appointed as a Community Reference Laboratory (CRL) for mycotoxins in animal products. This chapter encompasses both human food and animal feeds, and since humans consume meat, which may be contaminated through animal feeds, the word feed is not used in the text. Toxicological aspects of only the most important and well-studied mycotoxins are described in the following sections.


Table 11.4 Mycotoxins Produced by Fusarium Species and Miscellaneous Fungi Mycotoxin Fusarium species toxins Deoxynivalenol, nivalenol T-2 toxin Fumonisins Moniliformin Zearalenone Trichothecenes Monoacetoxyscirpenol Miscellaneous fungal toxins Psoralens Ergotoxins Mushroom toxins Alternariol, alternariol monomethyl ether, tenuazonic acid

Producing organism F. graminearum, F. culmorum, F. crookwellense F. poae, F. sporotrichioides F. moniliforme, F. proliferatum Fusarium spp. F. graminearum, F. culmorum, F. crookwellense Fusarium spp. Fusarium roseum Myrothecium spp., Sclerotinia sclerotiorum Claviceps purpurea Amanita spp. Alternaria alternata, A. tenuis

Table 11.5

Natural Occurrence of Mycoxotins in Foods and Feeds

Toxin

Commodity contaminated

Alternariol Emodin Sterigmatocystin Sporidesmins Patulin

Penicillic acid Fumonisins Ochratoxin

Psoralens Citrinin Aflatoxin M1 Aflatoxin B1

T-2 toxin Vomitoxin Diacetoxyscirpenol Zearalenone

Pecans, sorghum Chestnuts Pecans, coffee Grass litter, grass Apples, apple cider, apple juice, pears, grape juice, bananas, pineapples, grapes, peaches, apricots Corn, dried beans Corn, corn-based foods and feeds Corn, barley, wheat, oats, rye, green coffee beans, beans, groundnuts, hay, animal products (pork and poultry), poultry feed, Celery, parsley Groundnuts, wheat, oats, barley, rye Milk, dried milk products, buffalo milk, yogurt, fresh and processed cheese Groundnuts, corn, groundnut oil, groundnut oilcake, peanut butter, cottonseed meal, pecans, pistachio, figs, Aleppo pinenuts, hazelnut, walnut, almonds, legumes Barley, corn, sorghum Corn, barley Feed corn, mixed feed Corn, corn flakes, sorghum, feedstuffs, sesame meal, hay, barley,

11.2 MYCOTOXINS OF ASPERGILLUS SPECIES 11.2.1

Aflatoxins

Aflatoxins are a group of extremely toxic metabolites produced by the common fungi Aspergillus flavus, A. parasiticus, and A. nominus. These fungi are ubiquitous and, under favorable conditions, can grow on a wide variety of agricultural commodities. Although A. flavus is associated with most food and feed contamination with aflatoxins, only a few strains of A. flavus actually produce this toxic metabolite. This mold is taxonomically related to the larger Aspergillus flavus-oryzae group, which is widely distributed, in the soil and in many foodstuffs (Concon, 1988). Historical Aspects Aflatoxins were first identified as etiological agents for animal disease in the early 1960s, after an outbreak of deaths of turkeys in England and elsewhere. The disease, termed turkey X disease because of the then-unknown


Reporting country United States United States Southwest Africa Australia, New Zealand United States, France, Germany, Canada, Sweden United States United States, India, China, South Africa United States, Canada, Denmark, Finland, France, Norway, Poland, Sweden, United Kingdom, Yugoslavia, Bulgaria, Japan United States, Italy Canada, India Iran, Germany, India, South Africa, United States, France Worldwide

Canada, United States, Russia, India United States, Japan, South Africa Germany, United States United States, Canada, Russia, India, Japan, South Africa, Germany, Yugoslavia, Scotland, France, Finland

cause, was characterized by acute hepatic necrosis, marked bile duct hyperplasia, acute loss of appetite, wing weakness, and lethargy (Blount, 1961). Similar symptoms and lesions in poultry were reproduced later (Asplin and Carnaghan, 1961). The cause of these effects was traced to the Brazilian groundnut meal that was used in a component of the poultry ration (Allcroft and Carnaghan, 1963). These effects were not confined to poultry: in other animals that consumed rations containing groundnut meal exported from African countries similar pathologic signs concurrently developed (Loosmore and Markson, 1961). An intense investigation into the cause of these outbreaks then began. The causative agents as well as the responsible fungal species were rapidly identified by several workers (Nesbitt et al., 1962; Sargeant et al., 1963). The identified compounds were named aflatoxins after one of the fungi from which they are produced, Aspergillus flavus, and were given names descriptive of their metabolites relative to the solvent front on thin-layer chromatography (TLC) plates as well as their fluorescent color. The outbreak of turkey X disease, clearly described and documented, had been preceded by a number of less

well-described episodes of epizootics in a number of animal species (Paget, 1954; Schoental, 1961; Burnside et al., 1957). Thus, aflatoxins appear to have existed for a considerable time before the epizootic outbreak in England in 1960. However, that dramatic episode of the hepatotoxic disease, which initially destroyed more than 100,000 turkeys, demonstrated the seriousness of the problem facing the food animal industry and ultimately led to the recognition that aflatoxin is both an economic and a public health problem in many areas of the world. Interest in the health effects of aflatoxins might have been confined to the veterinary establishment had it not been for the fact that groundnut meal was being considered by the World Health Organization as a protein supplement for malnourished children in third world countries (Stoloff, 1977). Equally important were reports that aflatoxins were not only acutely toxic, but also carcinogenic in experimental rats (Schoental, 1961; Dickens and Jones, 1963a). As more commodities, such as corn, cottonseed, and rice, were discovered to contain aflatoxins, the potential health problems surrounding these mycotoxins generated much wider concern. Structural Diversity and Chemical Characteristics Aflatoxins consist of a group of approximately 20 related fungal metabolites. They are produced by Aspergillus flavus, A. parasiticus, and A. nominus and can occur in a wide range of important raw food commodities such as cereals, nuts, spices, figs, and dried fruit. Originally, the toxic factors isolated from feed were separated chromatographically into four distinct compounds: aflatoxins B1, B2, G1, and G2 (Figure 11.1) (Nesbitt et al., 1962; Sargeant et al., 1961). The molecular formulae indicated that aflatoxins B2 and G2 (AFB2 and AFG2) were dihydro derivatives of the parent AFB1 and AFG1, respectively (Nesbitt et al., 1962; Cheung and Sim, 1964; van Soest and Peerdeman, 1964). AFB1 and AFB2 fluoresce blue under UV light with RF values of 0.4 and 0.36, respectively, on thinlayer silica plates developed in chloroform-methanol. The other two, with slight lower RF values of 0.34 and 0.31, fluoresce turquoise green and are designated AFG1 and AFG2, respectively. Aflatoxins contain a coumarin nucleus fused to a bifuran and either a pentanone (AFB1 and AFB2) or a sixmembered lactone (AFG1 and AFG2). AFB1 and AFG1 were more toxic to ducklings, rats, and fish than either AFB2 or AFG2; AFB1 was the most toxic (Wogan et al. 1971; Abedi and Scott, 1969). A similar pattern holds for its carcinogenic potency, AFB1 > AFG1 > AFB2 (Wogan et al., 1971; Ayres et al., 1971).


Allcroft and Carnaghan (1963) reported that extracts of milk from cows fed aflatoxin-containing groundnut meal, when administered to ducklings, induced liver lesions that were identical to those caused by AFB1. However, extracts of toxic milk by TLC showed no AFB1 (the main aflatoxin in the groundnut meal) present. Subsequently, DeIongh and associates (1964, 1965) showed by silica gel TLC that the toxic factor was a blue-violet fluorescing compound that had an RF value well below that of AFB1. They produced chromatographic evidence that the lactating rat was able to convert AFB1 into this “milk toxin” and also that the factor was produced by cultures of A. flavus on crushed groundnuts. Allcroft and coworkers (1966) later suggested the trivial name aflatoxin M for this milk toxin. Further chromatographic studies in different solvent systems resolved this milk toxin into two fluorescing components, a blue-violet one of RF 0.34 and a violet one of lower RF value (0.25). These were subsequently designated aflatoxins M1 and M2, respectively (Figure 11.1). AFM1 and AFM2 are hydroxylated aflatoxin derivatives, which were subsequently isolated from lactating rats, rat liver, and sheep urine, liver, and kidneys, as well as from moldy groundnut and corn. AFM1 was established to be as toxic as but less carcinogenic than AFB1 (Purchase, 1967; Canton et al., 1975). AFGM1 and AFGM2 (Figure 11.1), the hydroxylated derivatives of AFG1, were isolated from A. flavus cultures (Heathcote and Dutton, 1969). However, these are very minor natural metabolites. The 2-hydroxyaflatoxins (Figure 11.1) produced by A. flavus have been reported under several names by various scientists. They are known as AFB2a and AFG2a (Dutton and Heathcote, 1966, 1968) but have also been described as AFB1 hemiacetal (Buchi et al., 1966; Pohland et al., 1968), aflatoxin-W (Andrellos and Reid, 1964), and hydroxydihydroaflatoxin B1 (Ciegler and Peterson, 1968). AFB2a and AFG2a are relatively nontoxic (Dutton and Heathcote, 1966). In view of the susceptibility to hydration of the 2-3 double bond in the terminal furan ring of aflatoxins B1 and G1, the theoretical possibility arose that both aflatoxins M1 and GM1 could be hydrated in dilute acid to form the corresponding hemiacetals. The hemiacetal of AFM1 might also be formed in vitro by liver homogenates. Subsequently, these two new aflatoxins were isolated and characterized, were found to be dihydroxy aflatoxins, and were designated as M2a and GM2a. A possible precursor in the biosynthesis of aflatoxins was isolated from rice and wheat inoculated under the laboratory conditions. It was described as 6-methoxy-7(2′-hydroxyethyl)difurocoumarin and named parasiticol (Stubblefield et al., 1970) or AFB3 (Figure 11.1) (Heath-

O

O

O O

O

O

O

O O

O

OH

OCH3

O

O

OCH3

O

Aflatoxin B1

O

O

O

O

O

O

OCH3

O

O

O

O

O

O

O

OCH3

Aflatoxin M2a

HO

O

O

O

O

O

O

O

O

HO

O

Aflatoxin GM2a

O

OCH3

O CH2

O

O

Aflatoxin B2a

O

OCH3

Aflatoxin GM2

O

CH2OH

OCH3 Aflatoxin B3

Figure 11.1 Chemical structures of naturally occurring aflatoxins B1, B2, G1, G2, M1, and M2, and their metabolites.


O

O

O

OH

OCH3

O

OCH3

O

O O

O

O

OH

O

Aflatoxin GM1

O

O

OH

O

Aflatoxin G2

Aflatoxin B2

HO

OCH3

OCH3

Aflatoxin M2

O O

O

O

Aflatoxin M1

OH

O

O

OCH3

O

O

OH

O

Aflatoxin G1

O

O

O

HO

O

O

OCH3 Aflatoxin G2a

cote and Dutton, 1969). AFB3 is a natural metabolite of A. flavus and A. parasiticus. Rhizopus species are also capable of metabolizing AFG1 to form AFB3 (Cole and Kirksey, 1971). Ducklings appear to be as sensitive to AFB3 as to AFB1, whereas AFB3 is much less toxic to chick embryos than AFB1 (Stubblefield et al., 1970). Several important metabolites of aflatoxins have been identified. Reduction of the pentanone of AFB1 by microorganisms yields aflatoxicol (AFL or AFR0) (Figure 11.2) (Detroy and Hesseltine, 1970). The importance of AFL increased dramatically when it was demonstrated to be produced by animals and its highly toxic and carcinogenic activities became apparent (Patterson and Roberts, 1971; Schoenhard et al., 1974). Animals also metabolize AFB1 by O-demethylation to produce AFP1 (Figure 11.2). It has been detected in the urine of several animal species (Palmgren and Ciegler, 1983). AFP1 is less toxic than AFB1 . In vitro metabolism by liver microsomes from monkeys and humans produces a major metabolite of AFB1, AFQ1. It contains a hydroxyl group, which is in the β-

O

OH

O

H

position to the carbonyl group of the cyclopentanone-ring in AFB1 (Figure 11.2) Detoxification procedures using ammonia produce two major derivatives of AFB1: AFD1 and AFD2 (Lee et al., 1974; Cucullu et al., 1976). The six-membered lactone ring in AFB1 is disrupted by treatment with ammonia (Figure 11.3). Physical and Chemical Properties Some of the physical and chemical properties of various aflatoxins are summarized in Table 11.6. They are crystalline substances, freely soluble in moderately polar solvents such as chloroform, methanol and dimethyl sulfoxide, and they dissolve in water to the extent of 1 to 20 µg/ml (Watson, 1998). They fluoresce under UV irradiation, although AFB1 and AFG1 need derivatization to enhance their fluorescence to a level similar to that of AFB2 and AFG2. This forms the basis for their detection by TLC or high-performance liquid chromatography (HPLC). On TLC plates the four substances are distinguished on the basis of their fluorescent color: B stands for blue and G for green or turquoise, and subscripts relate to their chromatographic mobility. AFB1 is usually found in the highest concentration. Crystalline aflatoxins are extremely stable in the absence of light and particularly UV radiation, even at tem-

Aflatoxicol (AFL or AFR0) O

O

O

OCH3

OH O

O

O

O

Aflatoxin P1

O

OCH3

Aflatoxin D1 O

O

OH

OH O

O

O H

O

Aflatoxin Q1

O

OCH3

OH O

O

OCH3

Aflatoxin D2

H

Figure 11.2 Some important metabolites of aflatoxins.


Figure 11.3 Proposed major reaction products from the ammoniation of aflatoxin B1.

Table 11.6 Physical and Chemical Properties of Some Aflatoxins Ultraviolet absorption maxima in methanol, ε Aflatoxin B1 B2 G1 G2 M1 M2 Aflatoxicol

Molecular formula

Molecular weight

Melting point, °C

265 nm

360–362 nm

C17H12O6 C17H14O6 C17H12O7 C17H14O7 C17H12O7 C17H14O7 C17H14O6

312 314 328 330 328 330 314

268–269 286–289 244–246 237–240 299 293 230–234

12 400 12 100 9 600 8 200 14 150 12 100 (264) 10 800 (261)

21 800 24 000 17 700 17 100 21 250 (357) 22 900 (357) 14 100 (325)

peratures in excess of 100°C. Solutions prepared in chloroform or benzene are stable for years if stored in a cold and dark place. The purity and concentration of reference solutions can be calibrated by using molar absorptivity data (Scott, 1990). The lactone ring makes aflatoxins susceptible to alkaline hydrolysis, and processes involving ammonia or hypochlorite have been investigated as means for their removal from food commodities. However, questions concerning the toxicity of the breakdown products have restricted the use of this means of eradicating aflatoxins from food and animal feeds. If alkaline treatment is mild, acidification reverses the reaction to re-form the original aflatoxin. AFB1 and AFG1 are converted to AFB2a and AFG2a by acid catalytic addition of water across the double bond of the furan ring. Oxidizing reagents react, and the molecules loss their fluorescence.

Representative sampling is of utmost importance in aflatoxin determination. Many mycotoxins are very inhomogeneously distributed in the commodities to be inspected, with the result that it is very difficult to draw a representative sample. Samples are further reduced in size to obtain test portions that usually vary in weight from approximately 20 to 100 g, a range resulting from a compromise between homogeneity requirements and practical considerations. Test portions then undergo the further steps outlined in Figure 11.4. All analytical procedures include three steps: extraction, purification, and determination. Extraction is usually

Sampling

Extraction

Analytical Methods The major aflatoxins of concern are B1, B2, G1, G2, M1, and M2; AFB1 is the most potent of these six naturally occurring aflatoxins. Because of the diversity of toxicological manifestations and the economic losses caused by exposure to aflatoxins, humans and susceptible animals must be protected from undue exposure to these toxins to safeguard their health. Numerous chemical and biological methods for the detection and quantification of AFB1 and related compounds have been proposed since the 1970s. The basic steps in aflatoxin and other mycotoxin determinations are outlined in Figure 11.4. The early observations of Sargeant and colleagues (1961), concerning the chemical properties of toxic components in suspected groundnut meal, form the basic technology for the separation and detection procedures, an extraction of aflatoxins with chloroform and detection of their fluorescence under UV exposure.


Cleanup

Concentration

Ultimate separation

Detection and quantitation

Confirmation of identity

Figure 11.4 Basic steps involved in the analytical procedures for mycotoxin determination.

done with (combinations of) organic solvents and water. The most common system for extraction has been a mixture of chlorohydrocarbon (e.g., chloroform) and water. This system is gradually being replaced by the methanolwater or acetonitrile-water system. Purification of the extract to remove lipids and other substances is usually done by passing the extracts through chromatography columns or prepacked cartridges (SepPak, Bond-Elut, Aflatest, Multifunctional, etc.). The latter are commercially available with many types of adsorbents and in many formats that may suit the needs of the analyst. The most recent advance in cleanup of extracts containing mycotoxins is the use of immunoaffinity cartridges. These columns are composed of monoclonal antibodies, specific for the toxin of interest, which are immobilized on Sepharose and packed into small plastic cartridges. These can be incorporated in fully automated sample preparation systems that take the sample from the extraction stage through to completion of HPLC. In addition to HPLC, other chromatographic techniques can be used for the ultimate separation and quantitation: TLC and gas-liquid chromatography (GLC). TLC was very popular in the 1960s and 1970s; though still very valuable and widely applied in the developing countries, it was largely superseded during the 1980s by HPLC. The latter is an attractive alternative to the TLC fluorodensitometry and visual analytical procedures, because of its high resolution potential, rapidity of separations, and potentially improved quantitative accuracy and precision. Its limitations are that large numbers of samples cannot be quantitated as rapidly as with TLC and that interfering substances contained in extracts obtained from naturally contaminated foods and feeds impede the measurement of aflatoxins. However, for the detection of aflatoxins in smaller quantities of urine, blood, or other tissue samples, HPLC offers distinct advantages. GLC has limited applications in mycotoxin analysis because it requires volatile components, whereas most mycotoxins are nonvolatile. Besides the chromatographic techniques, the immunoassay techniques are worth mentioning. In particular, enzyme-linked immunosorbent assay (ELISA) has become an important technique in mycotoxin methodology (Deshpande, 1995). The simplicity of the ELISAs and the large number of samples that can be handled in 1 day have made these tests important, especially for screening and semiquantitative determinations. Their lack of selectivity might prevent their use as quantitative tools, and therefore they are less suitable for regulatory analysis. Another point that requires attention is their specificity. Although most of the antisera seem to be quite specific, the possibility of cross-reactions cannot be ruled out fully. It is therefore a good laboratory practice to confirm positive findings ob-


tained with immunoassays by using methods of analysis based on other principles. Currently, certified reference materials have become available for AFM1 in milk powder; for aflatoxins B1, B2, G1, and G2 in peanut butter; and for AFB1 in peanut meal and compounded animal feed. Many methods of analysis for aflatoxin in foods and feeds are published each year. An appropriate method must be selected for each need. Factors that should be considered before selecting a method include number of analyses needed, time, location, cost, equipment, safety, waste disposal, and, above all, the experience of the analyst. The simple, specific, and rapid immunoassays will play an increasingly important role in monitoring foods and feeds for aflatoxins and other mycotoxin contamination. Currently, these methods appear to have their greatest value when used in conjunction with existing TLC and LC methods. Biosynthesis Similarly to many other toxic secondary metabolites produced by fungi, aflatoxins are synthesized by the polyketide route, wherein head-to-tail condensations of acetate units proceed via poly-β-keto-thiol ester intermediates (Applebaum and Marth, 1981). In this biosynthetic pathway, the chain is initiated by acetyl CoA, and malonyl CoA is the source of additional carbon units. Relative to that of other polyketide-derived mycotoxins, the synthesis of aflatoxins has been particularly difficult to elucidate. It is now known that aflatoxins are derived from a C 20 polyketide (Coulombe, 1991). In the biosynthetic scheme for AFB1 (Figure 11.5), the initial intermediate is norsolorinic acid, which is converted to averufin in a two-step process via the intermediate averantin. The next biosynthetic step involves the ring opening of averufin, followed sequentially by dehydration, epoxidation, and epoxide rearrangement to form versicolorin A via versiconal A hemiacetal acetate. Versicolorin A is converted to the last major intermediate, sterigmatocystin, via an oxidoreductase. The o-methoxy group common to nearly all of the aflatoxins arises from the methyl donor S-adenosylmethionine (SAM) by catalysis of S-adenosyl methyltransferase. It converts sterigmatocystin to O-methylsterigmatocystin (Coulombe, 1991). The formation of the bisfuran ring system present in versicolorin A, sterigmatocystin, and AFB1 appears to involve the hydrolysis of the ester bond in versiconal hemiacetal acetate with elimination of an acetyl group, followed by ring closure. This proceeds via oxidation of the terminal hydroxyl group to the aldehyde, resulting in the hemiacetal structure.

OH O

O

OH

O O

10 CH3COOH

O

O OH

O

O

O

O

H 3C O

OH

HO

O O

CYCLIZED DECAKETIDE

OH

O

OH

OH

O

NORSOLORINIC ACID

OH

O

OH

CH3

H3C

AVERANTIN

O

AVERUFIN

OH

HO

OH

O

OH

O

O

OH

OH

O

OH

O

H3C

O

HO

OH

O O

VERSICONAL HEMIACETAL ACETATE O

O

OH

O

VERSICOLORIN A

O

O O

O

OH O

O

O

OCH3

AFLATOXIN B3

Figure 11.5 Proposed biosynthetic scheme for aflatoxin B1.


O

O

STERIGMATOCYSTIN

OCH3

The biosynthesis of other fungally-produced aflatoxins as well as their metabolic relationship are less clear and often the subject of conflicting reports in the literature. The formation of the tetrahydrobisfuran AFB2 and the dihydrobisfuran AFB1 probably arises independently by a branched-chain pathway beginning at versiconal hemiacetal through versicolorin A and versicolorin C, respectively. AFG1 and AFG2 probably also arise independently by a branched-chain pathway. There is good evidence that AFM1 and AFM2 are synthesized from the analogous B toxins via monooxygenase enzymes (Dutton et al., 1985). That many filamentous fungi can enzymatically hydroxylate tertiary carbon atoms is well documented in the literature (Sharma and Salunkhe, 1991).

Table 11.7 Enzyme Systems and the Types of Biotransformation Reaction Involved in the Metabolism of Aflatoxin B1

Biotransformation

AFQ1

As described in Chapter 5, biotransformation plays an important role in the biological activity and disposition of aflatoxins. Bioactivation of aflatoxins is a necessary step in the most dramatic of their toxic and carcinogenic effects. Several detoxification mechanisms involving biotransformation are also known. AFB1 represents by far the most toxic of the aflatoxins, and almost all of the available information on the bioactivity of aflatoxins in animals has focused on AFB1 and its metabolites. It is also usually the aflatoxin found in the highest concentrations in contaminated food and feed. Therefore, most of the discussion that follows focuses on AFB1; other aflatoxins are discussed whenever possible. For a detailed treatment of this topic, the readers are referred to several excellent reviews (Heathcote and Hibbert, 1978; Neal, 1987; Coulombe, 1991; Eaton et al., 1994; Watson, 1998). An overall scheme for the primary metabolism of AFB1, based on results obtained from in vitro studies using hepatic subcellular fractions isolated from a range of animal species, is shown in Figure 11.6. Not all metabolites have been identified in all species, and significant quantitative differences in the formation of the various products may exist. The enzyme systems and the types of biotransformation reaction involved in the metabolism of AFB1 are summarized in Table 11.7. A requisite step in the toxic and carcinogenic action of AFB1 is its conversion to one or more metabolites in various tissues of exposed animals. As in the case of other “procarcinogens,” the majority of metabolic conversions of AFB1 are catalyzed by cytochrome P-450s, which are a group of mixed-function oxidases present in the liver and other tissues. AFB1 is also transformed in various tissues by cooxidation reactions via prostaglandin H synthetase, although this pathway is generally of lesser importance


AFB1 metabolitea AFB2a

AFP1

AFM1

AFL

AFL-M1

AFL-H1

AFB1-epoxide

AFB1 dihydrodiol a

Enzyme systems and the type of reaction Microsomal metabolite of AFB1, doubtful enzymatic formation, occurs nonenzymatically through hydration of furan double bond in absence of cofactors Mixed-function oxidase–catalyzed odemethylase reaction in microsomes, major urinary metabolite in monkeys Hydroxylated metabolite, NADPHdependent mixed-function oxidase, major metabolite in milk and urine of animals fed AFB1-contaminated diets NADPH-dependent hepatic microsome– mediated hydroxylation of AFB1, major metabolite produced by primate microsomal metabolism Reversible reduction of AFB1 by reductase in the cytosol fraction, NADPH required as a cofactor, major metabolite of avian species Cytosol-catalyzed reduction of AFM1, or microsomal mixed-function oxidase–catalyzed hydroxylation of AFL Cytosol-catalyzed reduction of AFQ1 or by cytochrome P-450-catalyzed hydroxylation of AFL, major metabolite of AFB1 by humans or rhesus monkey Not isolated from biological systems or synthesized chemically, formation deduced from production of AFB1dihydrodiol as acid hydrolysis product of metabolically or chemically generated AFB1–nucleic acid adducts Formed by enzymatic or nonenzymatic hydrolysis of AFB1-epoxide

AF, aflatoxin; NADPH, reduced nicotinamide-adenine dinucleotide phosphate.

(Coulombe, 1991). It has long been known that species susceptibility to the effects of AFB1 depends, in large part, on the metabolic fate of this compound after exposure. Most of the metabolites shown in Figure 11.6 are less toxic than AFB1, whereas one possesses more toxicity. From a toxicological viewpoint, the most important reputed toxic intermediate of AFB1 is the AFB1-2,3-epoxide (or the 8,9-epoxide in IUPAC nomenclature). The epoxide undergoes rapid hydrolysis to form AFB1-2,3-dihydrodiol (and its Tris adduct, if this buffer is present) (Lin et al., 1978; Neal et al., 1981). AFB1-2,3-dihydrodiol can exist in a resonance form as a phenolate ion that is capable of

O

OH

O

Glucuronide conjugate

O

Aflatoxicol H1

O OH O

OH O

O

Aflatoxin M1-P1

O

O

O

OH

O

O O

O

Aflatoxin Q1

O

Aflatoxin P1

OCH3

O

Glucuronide conjugates

O

Aflatoxin M1

OH

O

O

O OH O

OH O

O

O

O

OCH3

O

O

OCH3

DNA adducts O O

O

OCH3 O

AFLATOXIN B1

O

OH

Aflatoxicol M1 O

GSH conjugate

OH

O

Aflatoxin B1-2,3-epoxide

O O O

O

O O

Protein adducts O

O

OCH3

OCH3 OH

O O

O O

O

Glucuronide conjugates

HO O HO HO

O

O

OCH3

O

O

Aflatoxin B2a

Aflatoxin B1-2-3-dihydrodiol

Figure 11.6 Overall scheme for the primary metabolism of aflatoxin B1 in animals.


OCH3

O Aflatoxicol

OCH3

forming Schiff base adducts with protein amino groups, particularly lysine (Figure 11.7). The diol is most likely to be involved in the binding of AFB1 to protein observed in vivo (Garner et al., 1979; Appleton et al., 1982). The AFBlysine adduct is the principal adduct found in plasma albumin after in vivo AFB1 exposure (Sabbioni et al., 1987). This type of reaction could be involved in the mechanisms of acute toxicity of aflatoxin. It is the metabolite responsible for alkylation of cellular nucleic acids and subsequent carcinogenic and mutagenic activity. Only a very small portion of administered AFB1 is generally present in the unmetabolized form in either the tissues or secretions of animals. The major hydroxylated metabolites of AFB 1 formed by cytochromes P-450 are AFM1, AFP1, AFQ1, and AFB2a (Figure 11.6, Table 11.7). Additional metabolites, which are generally formed in smaller quantities, depending on various conditions, include AFL M1 and AFL H1. These stable metabolites are considered to be detoxified relative to AFB1, are more polar, and, as such, are

O

O

O HO

O

HO

OCH3

O

AFB1-2,3-dihydrodiol

O O

O

H

H

O

O HO

-

O

OCH3

Phenolate resonance form

Schiff base formation with protein amino groups

Figure 11.7 Schiff base formation between protein amino groups and aflatoxin B1-2,3-epoxide.


more easily excretable. The cyclopentanol AFL is not a product of oxidative metabolism, but rather a result of the reductive metabolism of AFB 1 catalyzed by soluble NADPH-dependent reductases (Wong and Heish, 1978). For the secondary metabolism (phase II reactions, see Chapter 5), the formation of sulfate and glucuronic acid conjugates of AFB1, and a range of primary microsomal metabolites (phase I products) of AFB1, including AFP1, AFM1, and AFQ1, have been described (Wei et al., 1978). Sulfate or glucuronide conjugates are excreted in the urine and bile of animals treated in vivo with AFB1 (Dalezios et al., 1971; Bassir and Emafo, 1970). AFB1 epoxide can be conjugated with reduced glutathione, a process requiring the presence of the appropriate glutathione S-transferase (GSTase) enzymes. The glutathione conjugate has been chemically characterized and its possible role in modifying AFB1 toxicity described (Degen and Neumann, 1978; Moss et al., 1983). The subsequent stages in the mercapturic acid pathway have been examined in in vitro systems (Figure 11.8). The initial stage is catalyzed by γ-glutamyl transpeptidase, which is particularly abundant in the brush borders of the proximal tubules of the kidney. Examination of bile of AFB1-treated rats indicates that the majority of AFB1 conjugated with glutathione (GSH) in the liver emerges into the common bile duct in the form of AFB1-GSH conjugate, despite the presence of high levels of γ-glutamyl transpeptidase on the canalicular surfaces of the bile ducts through which the AFB 1-GSH conjugate is secreted (Moss et al., 1984). When a large proportion of the liver hepatocytes contain elevated amounts of γ-glutamyl transpeptidase, as a result of feeding hepatocarcinogenic substances such as AFB1, the conjugated AFB 1 in the bile primarily comprises AFB1-Cys-Gly. The subsequent stages in the formation of the AFB 1 -mercapturate, dependent on the activity of dipeptidase and N-acetyl transferase, have been demonstrated in vitro in rat kidney–derived systems (Moss et al., 1985). The mercapturate of AFB1 has been detected in the urine of rats and marmoset monkeys after the acute administration of AFB1. The fate of AFB1 is dependent on the relative activity of several biotransformation pathways, in addition to other factors such as DNA repair rates (Figure 11.9). The amount of the mycotoxin that exerts carcinogenic or toxic effects depends on the amount converted to various metabolites as well as on the biological activity of those metabolites. With respect to carcinogenicity, AFB1-2,3-epoxide is the key active metabolite. As indicated in Figure 11.9, hydroxylated metabolites of AFB1 (AFM1, AFP1, AFQ1) are assumed to represent detoxification products. Detoxification of the reactive epoxide also may occur through conjugation with GSH. Hydrolysis of AFB1-2,3-epoxide forms

Aflatoxin B1-2,3-epoxide O

O

O O

O

Macromolecular binding

Microsomal mixed-function oxidase O

O

O

O O

OCH3

AFLATOXIN B1

O

OCH3

Epoxide hydratase

Glutathione S -transferase GSH

AFB1-2,3-dihydrodiol

AFB1-N-acetylcysteine O CH3 CO

O

O

O COOH HO

O

CHCH2CH2CONH

O

NH2

CHCH2S

O

O

OCH3

HO CONHCH2COOH

NH CHCH2S

O

O

AFB1-GSH

OCH3 Glycylglycine

COOH γ-Glutamylglycylglycine Acetyl CoA

γ-Glutamyltranspeptidase

N -Acetyltransferase O

O

O

O

CoA O

O OH

OH

Dipeptidase

NH2 CHCH2S COOH

O

O

OCH3

AFB1-Cys

Figure 11.8 Mercapturic acid pathway of phase II biotransformation of aflatoxin B1.


NH2 CHCH2S

O

CONHCH2COOH

O

OCH3

AFB1-Cys.Gly

AFLATOXIN B1 Hydroxylated Metabolites

Epoxide

GSH Conjugate Dihydrodiol

Protein Binding

TOXICITY

DNA Products

CANCER

Activation products

Detoxification products

Figure 11.9 Schematic representation of the role of various biotransformation pathways in the disposition, toxicity, and carcinogenicity of aflatoxin B1.

a dihydrodiol that probably still is capable of causing toxic effects via binding to proteins but presumably is a less potent carcinogenic species than the epoxide. The metabolism of the aflatoxins other than AFB1 has received rather less research interest because of their lower potencies and rate of occurrence. The metabolic pathways described in the literature for AFG1 are shown in Figure 11.10. The doubt concerning the metabolic formation of AFB2a from AFB1 applies also to the formation of AFG2a from AFG1 (Patterson and Roberts, 1970). The formation of a 4-hydroxylated AFG1 is catalyzed by microsomal mixed-function oxidases (Patterson, 1973). AFG1 is both acutely toxic and carcinogenic, and the proposed mechanism of metabolic activation, epoxidation of the terminal furan ring, similar to the proposed mechanism of activation of AFB1, is based on chemically and metabolically catalyzed formation of DNA adducts (Garner et al., 1979). The principal pathways reported for the metabolism of AFB2 by animal species are given in Figure 11.11.


Lacking the double bond in the terminal furan ring, and given the apparent importance of this feature in the metabolic activation of AFB1 and AFG1, it is of some interest to account for the albeit rather lower, but nevertheless well-authenticated toxicity of AFB2 (Wogan et al., 1971). AFB2 is converted to AFB1 in both rat and duck liver, and the rates of these reactions appear to correlate with the toxic potential of AFB2 in these species. The formation of AFB2a from AFB2 cannot be accounted for in terms of artifactual hydration, as in the case of AFB1 and AFG2a from AFG1, and presumably represents a direct hydroxylation of AFB2. Hydroxylation of AFB2 producing AFM2 and reduction of the cyclopentenone carboxyl forming dihydroAFL has also been described (Patterson, 1973). It is important to note that not all of the biotransformations occur in every species and that the profile of metabolites formed from AFB1 varies with species, tissue type, and age of the animal. Many of the same dietary factors or agents known to modify the metabolism of other

O

O

O

O

OH

O

O

OCH3

AFGM1

O

O

O

O O

O

O

O

O O

O

OCH3

O

AFG1

OCH3

AFG1-2,3-epoxide

O

O

O

HO

O

O

O

O

OCH3

AFG2a

Figure 11.10

Pathways of aflatoxin G1 metabolism in animal systems.

carcinogens similarly affect AFB1 biotransformation patterns as well as in vivo biological effects. The prior or concurrent administration of a wide variety of modulating agents, such as flavone and indole compounds, phenolic antioxidants, polyhalogenated hydrocarbons, phenobarbital, and 3-methyl-cholanthrene, as well as dietary factors, such as protein level, protein quality, and dietary fat consumption, are known to affect the metabolism of aflatoxins. Occurrence in Foods After the finding that aflatoxins were potent hepatocarcinogens, numerous surveys were carried out on the natu-


ral occurrence of these mycotoxins in foods. Among foods and foodstuffs, groundnut and groundnut products, which are widely grown and utilized for food and feedstuffs in tropical and subtropical countries, would seem to be significantly contaminated with aflatoxins. The contamination of groundnut and groundnut products with aflatoxins has been reported in the United States, Africa, Asia, the Indian subcontinent, as well as other countries. Aflatoxin has also been found in other kinds of edible nuts, including almonds, hazelnuts, and pistachio nuts, although the levels have been relatively low. Aspergillus flavus is able to infect several kinds of cereals, in particular corn, and produces aflatoxins before

O

O

O OH

O O

O

OCH3 AFM2

O

O

O

O

O

OCH3

O

O

AFB1

O

OCH3

AFB2

O

OH

O

O

O

O

O

O

OCH3

HO

Dihydro AFL

Figure 11.11

O

O

O

OCH3 AFB2a

Pathways of aflatoxin B2 metabolism in animal systems.

harvest. The levels and frequency of aflatoxin contamination are significantly high, and corn heavily contaminated with AFB1 may induce acute hepatitis in humans. AFB1 and related mycotoxins are often detected in cereals such as corn, barley, wheat, sorghum, oats, millet, rice, beans, cowpeas, peas, soybeans, sesame, sweet potatoes, cassava, and other local edible grains and foods (Stoloff, 1976; Newberne and Butler, 1969; Coulombe, 1991). Although their contents are low relative to the levels found in groundnuts and corn, these commodities are important foods worldwide. The aspergilli may infect these commodities in the field or in storage. Factors that promote the growth of this fungus include a relatively high water activity in the field (0.84 to 0.86), high temperature, drought stress, mechanical damage during harvest, insect infestation, rain during harvest, and moisture accumulation during storage (Stoloff, 1976). Drought may act to increase the susceptibility of groundnuts to aflatoxins by reducing the ability of the plant to produce phytoalexins (Dorner et al., 1989).


Drought conditions in the midwestern and southeastern United States in the late 1980s resulted in increased aflatoxin contamination in corn (Brosten, 1989). Dairy products are sometimes contaminated with AFB1 and AFM1. The consumption of AFB1-contaminated feeds by lactating animals results in the extraction of metabolites such as AFM1. The relative amount of AFM1 excreted is related to the amount of AFB1 in the feed, and about 0.1% of AFB 1 ingested is excreted into milk as AFM1 . Kiermeier and Mashalev (1977) reported that AFM1 was detected in 8 dried milk products of 166 samples tested in the range of 0.7–2 ppb, which corresponds to about 0.08–0.26 µg/L. Meat and meat products are also contaminated with aflatoxins when farm animals are fed with aflatoxin-contaminated rations. Furthermore, many meat products such as “country style” hams and certain sausages are traditionally mold-ripened. As suggested by Leistner (1984), undesirable Penicillium spp. and other fungi grow quite frequently on meat products, especially on fermented sau-

sages (salami) and raw hams. Natural inoculation has been the basis for the traditional methods, but now there is a trend to control the ripening process by using selected starter nontoxic strains. Aflatoxins have also been shown to occur in spices, including cayenne pepper, Indian chili powder, dried chili peppers, black pepper, capsicum peppers, and nutmeg (Jones 1977). Natural occurrence of aflatoxins in cereals and cereal products is summarized in Table 11.8. Toxicology Aflatoxins are both acutely and chronically toxic. AFB1, in fact, is one of the most potent hepatocarcinogens known (Fishbein, 1979). Hence, the long-term chronic exposure to extremely low levels of aflatoxins in the diet is an important consideration for human health. In the temperate, developed areas of the world, acute poisoning in animals is rare and in humans is now extremely unlikely. Extremely high levels of aflatoxins in the imported groundnut meal caused the outbreak of the turkey X disease in England. This alerted industry and governments to the potentially devastating effects of mycotoxins, particularly the aflatoxins. Acute Toxicity in Animals The original identification of the aflatoxins as important naturally occurring toxins resulted from their acutely toxic and carcinogenic effects in sensitive species fed contaminated feedstuffs. The acutely toxic properties were recognized as a result of the investigation into turkey X disease in domestic flocks in England caused by feeding of contaminated groundnut meal. The carcinogenic properties were detected by a subsequent experimental feeding of the contaminated meal to rats and also by recognition that outbreaks of hepatoma in rainbow trout were the result of feeding of cottonseed meal contaminated with aflatoxins. These early observations therefore highlighted the potential hazards of dietary exposure to levels of these toxins existing in naturally contaminated foodstuffs, which could provoke an acutely toxic reaction in sensitive species. The greater potential hazard from prolonged human exposure to the much lower levels, which could provoke a carcinogenic response in sensitive species, was evident. Since the outbreak of turkey X disease in the 1960s, recognition of sources of aflatoxin contamination and the factors affecting its production have received very extensive research attention, which has led to a general lowering of levels of contamination. However, samples of feedstuffs containing levels of aflatoxins in excess of 1 to 2 ppm are


still being identified (Neal, 1998). This level of contamination is capable of presenting a chronic hazard to susceptible species. There have been many experimental animal studies using aflatoxins, too numerous to detail in this review. The toxicity of aflatoxins has been demonstrated in many domestic and experimental animals. A common feature is its potent hepatotoxicity. In fact, liver is a target organ for toxicity in almost all species studied thus far. Toxic effects have also been reported for other organs, including kidney, lung, colon, myocardium, and nasal epithelium. One of the most important facts to emerge from the experimental animal studies has been the recognition of a wide species variation in sensitivity to these toxins. In the vertebrate species tested, there is at least a 10-fold variation in the susceptibility to the acute effects of AFB1, and no species tested thus far is totally resistant. Differences in age, sex, strain, and route of administration are important factors in the toxic potency of AFB1. For example, male rats are generally more sensitive than females of the same age, and both very young and old mice and rats are the most susceptible to AFB1 (Newberne and Butler, 1969; Hayes et al., 1977). Oral administration of AFB1 is more effective than intraperitoneal administration in PortonWistar rats, although the reverse is true in Swiss mice (Busby and Wogan, 1984). Species relatively resistant to acute AFB1 include the mouse and hamster, with oral LD50 values of 9.0 and 10.2 mg/kg, respectively. The duck, monkey, and rainbow trout are relatively sensitive, with values of 0.34 to 0.56, 3.0, and 0.81, respectively (Patterson, 1973; Bauer et al., 1969). Rats are not particularly sensitive to acute aflatoxicosis, and oral LD50 values vary from 1.2 to 17.9 mg/kg (Hayes et al., 1977; Patterson, 1973). Poultry are sensitive to acute AFB1, although chicks are more resistant than are ducklings and young turkeys. Patterson (1973) observed that the rates of AFB1 metabolism as well as the profile of metabolites dictate the relative susceptibility to acute aflatoxicosis. For example, sensitive species generally metabolize an LD50 dose of AF1 rapidly, and liver preparations from those same species generally were shown to convert AFB1 to AFL more efficiently. Animals exposed to AFB1 often exhibit malaise, loss of appetite, and lower growth rates. Histopathological effects due to acute AFB1 are most dramatic in liver and are observed in all vertebrate animal species exposed to AFB1. The principal hepatic lesions are hemorrhagic necrosis, fatty infiltration, and bile duct proliferation. The hepatic zone in which these lesions occur varies. In ducklings and adult rats, the periportal zone is most commonly affected; in pigs, guinea pigs, and dogs, centrilobular lesions predominate. Hemorrhagic necrosis has also been observed in

Table 11.8 Natural Occurrence of Aflatoxins in Selected Agricultural Commodities

Commodity

Country

Corn Yellow corn Corn White corn (feed grade) Corn Corn Corn Corn Corn Corn Corn Corn Wheat Wheat Wheat flour Spaghetti Sorghum Sorghum Oats Oats Barley Millet Millet Millet Rye Rice Rice Rice and rice products Boiled rice Rough rice Raw rice Rice Garri (manihot flour) Red pepper Yam flour Beans

United States United States United States Nigeria Nepal China Philippines Thailand Japan India Kenya Philippines United States France France Canada Uganda France Sweden France France Uganda Thailand Nigeria France Philippines Thailand Philippines Philippines Brazil Nepal

Cassava Yam Sweet potato Cocoa Pistachio nut

Philippines Philippines Philippines

Nigeria Nigeria Nigeria Japan

Japan

Source: Compiled from Ueno (1985, 1987).


Aflatoxins, µg/kg, ppb 3–27 4–308 10,000 100–1000 8.8–37.5 B1 89.5 400 93 B1 131–340 B1 6250–15,600 3200–12,000 45.9 2–19 0.25–180 0.25–150 13 1–1 000 0.25–100 2 600 0.25–100 0.25–10 1–100 248 1.4 0.25–100 16 98 30 0.6 400 5–10 40 1 600 700 400 1.3–26.9 B1 0.4–6.9 B2 467.5 88.8 60.6 50.6 2.0–800 B1 0.4–180 B2 0.6–51.4 G1 0.2–16.3 G1 1.8–39.3 M1

Remarks 2.7% Positive 34.6% Positive Associated with aflatoxicosis in chickens 100% Positive 51% Positive 36% Positive ( G1 > B2 > G2 (Wogan et al., 1971). Aflatoxins with an unsaturated terminal furan ring, such as AFB1 and AFG1, had similar acute potencies in rats and ducklings, whereas AFB2 and AFG2, which contain a saturated terminal furan, were much less potent in ducklings and were nontoxic to rats at a dose of 200 mg/kg. That AFG1 was less acutely toxic than AFB1 also indicated that substituents fused to the lactone ring are also involved in determining the toxicity of aflatoxins. Acute Toxicity in Humans That humans are sensitive to acute aflatoxin poisoning, aflatoxicosis, appears to be beyond doubt. Though epidemiological studies have suggested that aflatoxins may be responsible for human diseases, there are several welldocumented episodes of acute poisoning in humans. Much of the information available today indicates that human exposures to high levels of aflatoxins in food are much more frequent in third world countries whose climates and prevalent agricultural practices favor mold infestation of foodstuffs. Thus, most of the recorded outbreaks of acute aflatoxicosis have occurred in Southeast Asia and Africa. Campbell and Stoloff (1974) extrapolated from primate data the doses of AFB1 that would be required to cause acute aflatoxicosis in humans. Recognizing the uncertainties associated with species extrapolation, they predicted that persons who consume food containing 1.7 ppm for a short time could experience severe liver damage, that a single dose of 75 mg/kg could result in death, and that a daily dose of 0.34 mg/kg would not result in apparent acute aflatoxicosis. The involvement of aflatoxins in acute poisoning of humans is very well documented as a result of an episode in India. In two adjacent western states (Gujarat and Rajasthan), a food-borne toxicosis started during late October 1974 in rural areas where the staple food was corn. During this month, there were unseasonal rains, which drenched the standing corn crop; a total of 397 patients were affected and 106 died (Krishnamachari et al., 1975). Clinical features were characterized by jaundice, vomiting, and anorexia and followed by ascites, which appeared rapidly within a period of 2–3 weeks. The liver was enlarged and tender in only a few cases. Death was usually sudden and


in most cases preceded by massive gastrointestinal bleeding. Twice as many males as females were affected. Liver histopathologic analysis revealed extensive bile duct proliferation with periductal fibrosis. During the outbreak and for several weeks afterward, a large number of dogs exhibited ascites and icterus and died within 2–3 weeks of the onset. Affected corn grains obtained from afflicted households showed the presence of A. flavus, and chemical analysis revealed the presence of aflatoxins in the range 6.25–15.6 ppm. Since an adult consumes about 350 g of corn daily, the patients would have ingested 2–6 mg of aflatoxin daily for several weeks. These findings indicated that the hepatitis was due to the uptake of AFB1. A subsequent outbreak of fatal hepatitis in Kenya was also attributed to aflatoxin-contaminated corn (Ngindu et al., 1982). Several other case reports have also been reviewed in the literature (Campbell and Stoloff, 1974). Establishing causality in these situations is very difficult, but the circumstantial evidence, the measurably high levels of aflatoxins in food and human tissues, and the histologic findings are all persuasive. Aflatoxins have been implicated in subacute and chronic effects in humans. These effects include primary liver cancer, chronic hepatitis, jaundice, hepatomegaly, and cirrhosis through repeated ingestion of low levels of aflatoxin. Aflatoxins may also play an important role in causing a number of other human diseases, including Reye’s syndrome (Shank et al., 1971), kwashiorkor (Hendrickse et al., 1982; Hendrickse, 1991; Coulter et al., 1986), and hepatitis (Krishnamachari et al., 1975; Ngindu et al., 1982). Aflatoxins can also suppress the immune system (Pier, 1991). Mutagenicity As in the case of other procarcinogens, the binding of the AFB1-epoxide to cellular DNA is thought to be the initiating event in the AFB1-mediated mutagenesis and carcinogenesis. The level of AFB1-DNA adducts formed in a species or a tissue is often an accurate indicator of susceptibility to the carcinogenic effects of AFB1. Activated AFB1 binds exclusively with guanyl residues in DNA, and the AFB1-N7-Gua adduct is by far the most predominant form. Additional adducts have been isolated; of them the “ring-opened” derivative of the AFB1-N7-Gua adduct, the formamidopyrimidine, or AFB1-FAPyr, is the most common (Figure 11.12). The relevance of AFB1-DNA adduct formation to carcinogenesis has been the subject of intense study. The mutagenicity of AFB1 has been repeatedly demonstrated in systems employing bacteria (Lowery et al., 1983), yeast (Niggli et al., 1986), and human and other mammalian

O

O

O HO O

H2N

O

N

HN N

OCH3

O

N

AFB1-N7-GUA

O

O

O HO O N

HN H2N

N

O CHO

NH2

O

OCH3

AFB1-FAPyr

Figure 11.12 Structures of the major aflatoxin B1-DNA adducts. DNA, deoxyribonucleic acid.

cells (Billings et al., 1985; Kaden et al., 1987), among others. On a molar basis, AFB1 is one of the most potent mutagens known. In the Salmonella typhimurium test, it induces 8527 revertants per microgram, followed in potency by AFL (1940 revertants), AFG1 (285), AFM1 (275), AFL-H1 (170), AFQ (99), AFB2 (18), AFP1 (10), AFG2 (9), and AFB2a (2) (Wong and Hsieh, 1976). Carcinogenicity AFB1 is known to be carcinogenic in a wide variety of animals, such as the rat, mouse, duck, monkey, and rainbow trout. The major target organ involved after chronic exposure of AFB1 is the liver; tumors of other organs appear but are less prevalent. As is the case with acute toxicity, there exist significant species differences with respect to susceptibility. Other aflatoxins have proved to be less potent carcinogens than AFB1. The order of potency, as demonstrated in trout and rat feeding studies, generally follows that seen in acute studies from the same species. The approximate relative carcinogenic potency ranking follows


the order AFB1 > AFL > AFM1 >AFQ1 > AFG1; AFB2 and AFG2 are inactive (Ayres et al., 1971; Schoenhard et al., 1981; Hendricks et al., 1980; Coulombe, 1991). These studies again support the observations that the presence of an intact 2,3 double bond is a requirement for carcinogenicity and that substitution of the cyclopentanone ring of AFB1 to the dilactone of AFG1 results in a significant reduction in the biological activity of aflatoxins. The ecological process in which populations are compared in terms of average aflatoxin exposure and liver cancer rates has produced the most convincing evidence of an association between aflatoxins and hepatocellular carcinoma in humans. In some geographical areas, most notably sub-Saharan Africa, India, and Southeast Asia, primary hepatocellular carcinoma (PHC) is often the most common form of malignancy seen. In these areas, the consumption of aflatoxin-contaminated foods is also much more prevalent. In addition to tropical climates that favor growth of the fungus, harvesting, handling, and storage practices common in these regions, taken together, appear to promote widespread aflatoxin contamination. Several field studies conducted in Uganda (Alpert et al., 1971), the Philippines (Campbell and Salamat, 1971; Bulatao-Jayme et al., 1982), Thailand (Shank, 1977), Kenya (Peers and Linsell, 1973; Autrup et al., 1987), Mozambique (Van Rensburg et al., 1974), and Swaziland (Peers et al., 1976) have demonstrated a linear dose-response relationship between consumption of aflatoxin and human liver cancer. A high consumption of aflatoxin was consistently associated with a high incidence of PHC. Hsu and coworkers (1991) and Bressac and associates (1991) provided additional evidence of the role of AFB1 in human liver cancer. These researchers showed that 50% of human liver tumor tissue samples from regions in southern China and southern Africa associated with high dietary AFB1 level and PHC incidence contained a single point mutation in the tumor-suppressor gene p53. Confounding a firmer conclusion regarding the link between aflatoxin consumption and human PHC is the observation that hepatitis B virus (HBV) antigen is coincident in those geographical areas with high incidences of PHC. HBV infection is often assumed to be the major factor for PHC, since it has been demonstrated that the majority of PHC cases are characterized by chronic or previous HBV infection (Wild and Montesano, 1991; Neal, 1998). The extent to which the association between liver cancer and aflatoxin can be explained by an association between hepatitis B and aflatoxin is not clear. This relationship has been addressed in several studies (Peers et al., 1987; Yeh et al., 1989; Campbell et al., 1990) that, in general, conclude that an effect of aflatoxin in addition to hepatitis B is

functioning. According to Lutwick (1979), it is possible that AFB1 may act as cofactor to promote HBV-mediated chronic infection, cirrhosis, and liver cancer by suppressing cell-mediated immunity. More rigorous epidemiological studies on the role of AFB1 and human cancer may require the discovery of relatively HBV-free, high-dietaryaflatoxin populations, if such groups exist. A hypothetical scheme for the development of PHC is shown in Figure 11.13. The activation process of AFB1 mediated by the cytochrome P-450 system is regulated by genetic and dietary factors, and the reactive AFB1 epoxide attacks some promoter/regulation locus of HBV integrated into the host DNA. This leads to the expression of the protooncogenic property of HBV. Impairments of the immunosurveillance system by AFB1 and other mycotoxins such as trichothecenes, and tumor-promoting agents in foods, may cause the acceleration of tumorigenicity of the hepatic cells. AFB1 has been implicated as a factor in human liver cancer and classified as a probable human carcinogen by the International Agency for Research on Cancer (IARC). The current IARC classification for AFM1 is 2B (possibly carcinogenic to humans) (IARC, 1993). Teratogenicity Knowledge of the potential of mycotoxins for inducing birth defects has largely been obtained from laboratory

experimentation. In the initial study involving a mammal, Le Breton and associates (1964) reported that AFB 1 (300–400 µg per rat) given to pregnant rats caused increased prenatal death and hemorrhage at the uteroplacental junction. Fetal growth retardation followed chronic administration of smaller doses. Butler and Wigglesworth (1966) soon reported on the effects of oral aflatoxin doses of approximately 5.6 mg/kg on one of the gestation days 6–22 in Wistar rats. Animals treated before day 17 showed little effect, but fetuses from rats treated on day 17 were stunted. No malformations were reported, but the researchers described some fetuses as having “loose, wrinkled skin and unduly large heads.” Histological examination of fetuses and placentas yielded negative findings. When additional dams were treated with a more crude aflatoxin preparation, maternal liver damage was correlated with decreased fetal weight. Aflatoxin was detected in the fetal livers, however, so a direct toxic effect on the fetus could not be ruled out. Mammals other than the rat and hamster have been largely ignored in tests of aflatoxin teratogenicity. However, studies with nonmammalian species (e.g., chick embryos) have confirmed the toxicity of aflatoxins to developing systems. They also support the view that AFB1 is teratogenic only in specific organisms. Such data imply that AFB1 often displays neither specificity of action nor preferential concentration in specific tissues. At least one of these characteristics is probably necessary for the pro-

Hepatic cells HBV

(Integration into host DNA)

AFB1

Selection and hepatic injury (Activation) Mutation of protooncogenic HBV Dietary factors (Promotion) Immunosurveillance PRIMARY HEPATOCELLULAR CARCINOMA (PHC)

Figure 11.13

Multifactorial causation of aflatoxin-mediated primary hepatocellular carcinoma (PHC).


duction of malformations rather than the more general toxic effects, such as growth retardation. As a ready reference, the toxic symptoms of aflatoxins in various animal species are summarized in Table 11.9. Nutritional Modulation of Toxicity and Carcinogenicity Even before aflatoxins were isolated and identified as toxins and carcinogens, a dietary effect on their carcinogenicity had been reported. Investigators studying dietary deficiency of methyl group donors or lipotropes (the combined deficiencies of choline and methionine, sometimes with additional deficiency of folate) reported the appearance of hepatocellular carcinomas in deficient rats and much lower incidence in methyl-supplemented controls (Copeland and Salmon, 1946; Newberne et al., 1966). Both control and deficient diets contained groundnut meal, later shown to be contaminated with aflatoxins, to which the deficient rats were highly susceptible. The mechanism by which the deficiency of methyl group donors acts is not known, but several potential mechanisms have been demonstrated. Other dietary and nutritional effects on AFB1 toxicity and carcinogenicity in laboratory animals are known. High dietary levels (20% or more by weight) of corn oil increase AFB1 hepatocarcinogenesis in rats (Newberne et al., 1979). Extensive liver damage is seen in rats fed lowprotein diets (4%) compared to those fed 20% protein diets (Madhavan and Gopalan, 1965). The low-protein-diet animals had fatty livers, bile duct proliferation, and periductal fibrosis, whereas only minor changes were seen in the high-protein-diet group. Newberne and Wogan (1968) also found a higher incidence of liver tumors in a shorter time in rats that consumed a 9% protein diet than those fed 22% protein. Diets low in vitamin A tend to show a lower incidence of aflatoxin-induced liver cancer (Newberne and Rogers, 1972). Different dietary fats may also alter the potency of aflatoxins (Concon, 1988). AFB1 hepatocarcinogenesis is also invariably influenced negatively by increased intake of selenium in the diet (Lei et al., 1990). Some compounds, such as ethionine and the cyclopropenoid fatty acids and malvalic and sterculic acids from cottonseed, are cocarcinogens to aflatoxins (Newberne et al., 1966; Lee et al., 1966; Sinnhuber et al., 1966). In contrast, urethane, diethylstilbestrol (DES), and phenobarbitone are anticarcinogenic and reduce the tumor incidence or retard tumor development (Concon, 1988). Control and Detoxification Contamination of grains with mycotoxins results in substantial losses to agriculture (Stoloff, 1976; CAST 1989).


Every year a significant percentage of the world’s grain and oilseed crops is contaminated with aflatoxins. Unfortunately, discontinuing the feeding of aflatoxin-contaminated grain is not always practical, especially when alternate food and feedstuffs are not readily available or affordable. The prevalence of aflatoxins in a variety of foods destined for human consumption is a major concern. Interrelationships such as shown in Figure 11.14 exist among humans, animals, and aflatoxin-contaminated products. Detoxification should only be considered as a last resort, since the simplest and most practical way of reducing the contamination of foodstuffs by aflatoxins and related metabolites is to prevent the growth of those fungal species that produce them. Phillips and associates (1994) have grouped aflatoxin reduction strategies into the following five categories: 1. 2. 3. 4. 5.

Food and feed processing Biocontrol and microbial inactivation Structural degradation after chemical treatment Dietary modification of toxicity Reductions in bioavailable aflatoxin by selective chemisorption

Processing methods such as thermal inactivation, irradiation, solvent extraction and mechanical separation, density segregation, and adsorption from solution have been suggested for reducing aflatoxin content in foods. Aflatoxins are resistant to thermal inactivation and are not destroyed completely by boiling water, autoclaving, or a variety of food and feed processing procedures (Christensen et al., 1977). Aflatoxins may be destroyed partially by conventional processing procedures such as oil- and dry-roasting of groundnuts to be used as salted nuts, in confections, or in peanut butter (Lee et al., 1969; Marth and Doyle, 1979). A considerable reduction in aflatoxin levels has been associated with the limewater treatment (nixtamalization) of corn to produce tortillas (Ulloa-Sosa and Schroeder, 1969). However, subsequent studies have shown that much of the original aflatoxin is re-formed on acidification of the products (Price and Jorgensen, 1985). Although some destruction of aflatoxins has been achieved by conventional processing procedures, heat and moisture alone do not provide a very effective method of detoxification. Irradiation of contaminated products with shortwave and long-wave UV light and gamma irradiation has produced conflicting data. Although proven useful, solvent extraction methods for the detoxification of aflatoxincontaminated oilseed meals appear to be impractical and cost-prohibitive (Shantha, 1987). A new preharvest strategy for the prevention of aflatoxin contamination of groundnuts and cottonseed is the

Table 11.9 Toxic Effects of Aflatoxins in Various Animal Species Animal Poultry ducklings

Ducks

Coturnix quails Chicken

Turkey poults

Fish, rainbow trout Rodents, rats

Mice Guinea pigs Hamsters Ferrets Rabbits

Canines, dogs Ruminants, cattle

Sheep Primates, Rhesus monkey

Humans

Histopathology Extensive biliary proliferation in liver and fatty degeneration of peripheral parenchymal cells after intubation with 15 µg aflatoxin; proliferation of bile duct epithelial cells, vacuolation of focal parenchymal cells, slight to moderate fibrosis; liver infarction with 10–40 µg aflatoxin per day; mitochondrial necrobiosis and regressive changes, reticular fiber proliferation; hemorrhagic necrosis caused by aflatoxin M1 at high doses; at low doses, bile duct proliferation and extensive changes in the liver cells and renal tubular necrosis Decreased liver weights with sublethal doses of aflatoxin B1; renal parenchymal hemorrhage; liver atrophy, liver tumors with doses of aflatoxin B1; renal parenchymal hemorrhage; liver atrophy, liver tumor development after 14-month feeding; lowest tumorigenic dose is 0.03 ppm Bile duct proliferation, slight to moderate fibrosis, hepatic cell vacuolation Hydropic and fatty liver cells, cell vacuolation, slightly increased cell size, mild proliferation of ductules, hemorrhages in liver, muscle necrosis with diffused increase of sarcolemnal nuclei; decrease in RNA and vitamin A and fats with 10 ppm aflatoxin B1 for 3 weeks; decreased hatchability of eggs Liver, kidney, and myocardial congestion; duodenal catarrhal enteritis; retrogressive and regenerative changes in liver parenchyma; swollen liver cells; vacuolation in some cells; necrosis in perisinusoidal region; karyoorrhexis; karyolysis; grayish white surface and internal nodules throughout liver with fibrosis and bile duct proliferation Hyperplasia of bile duct epithelium and cholangitis; bile duct proliferation; hepatoma Liver enlargement, brownish yellow irregular nodular surfaces, red and greenish cysts, yellowish focal lesions, macroscopic grayish lesions with zones of hemorrhages in lungs; periportal liver necrosis; hyperplastic foci and preneoplastic lesions; atrophy of testicles, aspermatogenesis, retardation of fetal growth; teratogenesis; hepatocarcinoma, kidney tumors, carcinoma of glandular stomach; adenocarcinoma of colon; malignant sarcomas and fibrosarcomas at injection (subcutaneous) site Resistant to acute toxic effects even with high levels, but development of hepatomas, subcutaneous sarcomas; adenomatous pulmonary tumors Centrilobular necrosis, biliary proliferation within 48 hr; lesions in kidneys, adrenals, pancreas, and GI tract; severe edema and liver cirrhosis, hepatomas; kidney tubular reflux Liver lesions, teratogenesis Yellowish, hemorrhagic liver, fatty infiltration, centrilobular necrosis, cellular vacuolation in liver, bile duct proliferation, bile duct and hepatic cell tumors Lethal dose 65 ppm aflatoxin/kg body weight fed for 2 consecutive days; developed anorexia, retarded growth, weight loss, and death by fifth week of administration in 5-month-old rabbits at 40 µg/day Hepatic lesions, hepatitis like disease Severe tenesmus, liver fibrosis, ascites, visceral edema, centrilobular necrosis, ductal cell hyperplasia, occlusion of centrilobular veins; bile duct proliferation, chronic endophlebitis of centrilobular and hepatic veins, karyomegaly of some parenchymal cells, liver cirrhosis; epithelial nephritis and ulceration of abomasums; at levels of 0.7–1.0 µg aflatoxin/kg of feed decreased weight gains; gross evidence of liver damage at ≥ 0.7 µg/kg of feed, enlarged liver cell nuclei Relatively resistant, but hepatic parenchymal cell neoplasia, nasal carcinoma and nasal chondroma may develop; decreased fertility Fatty liver and cirrhosis; biliary fibrosis, severe fatty changes in parenchymal cells, soft enlarged livers, enlarged yellow kidneys with fat accumulation; viral hepatitis–like necrotic lesions; lethal to Macaca irus monkey at 50 µg aflatoxin B1/kg body weight Potent hepatocarcinogen, childhood cirrhosis, fatty infiltration of liver cells leading to cellular degeneration, fibrosis, hepatomegaly; acute poisoning lethal; acute hepatitis; Reye’s-like syndrome; subacute and chronic effects, e.g., primary liver cancer, chronic hepatitis, jaundice, hepatomegaly, cirrhosis; potent mutagen

Source: Modified from Concon (1988).


Animal waste

Compounded animal feed containing groundnut meal

4 µg Tc/cow/day

Cattle

0.02 mg/kg AFB1a Meat and meat products

Aspergillus flavus

1.4 ng/kg AFB1c Milk

Groundnut crop

Aflatoxin-contaminated nuts

0.07 µg/liter Mc

Groundnuts 0.015 mg/kg Tb

Human food

Storage

AFB1

Aflatoxin B1

a

U.K. Fertilizers and Feeding Stuffs Regulations

M

Aflatoxin M1

b

U.S. Peanut Industry Guidelines

T

Total aflatoxins

c

Approximate calculations

Figure 11.14

Schematic representation of the interrelations of humans, animals, and aflatoxin B1.

use of nontoxigenic strains of A. flavus and A. parasiticus fungi to compete with and exclude toxin-producing strains. Initial studies have shown that bioprevention can reduce preharvest aflatoxin contamination in these crops significantly (Cole et al., 1989; Cole and Cotty, 1990). Numerous chemicals, including acids, bases, aldehydes, bisulfite, oxidizing agents, and various gases, have been tested for their ability to degrade and inactivate aflatoxins. Although several have been found to be effective, most are impractical or potentially unsafe because of the formation of toxic residues or the perturbation of nutrient content, flavor, color, odor, texture, or functional properties of the product (Phillips et al., 1994). Two chemical approaches to the detoxification of aflatoxins that have received considerable attention are ammoniation and reaction with sodium bisulfite. The ammoniation process has been used successfully for many years in the United States, France, and Africa but has not yet been sanctioned by the U.S. Food and Drug Administration. Current research supports the use of ammoniation in an effort to reduce markedly the risk posed by aflatoxin contamination of grains and oilseeds. However, no safe


method currently exists to be used to eliminate the aflatoxin problem in the human food chain. 11.2.2

Sterigmatocystins

Sterimatocystin (ST) is a carcinogenic metabolite produced by fungi such as Aspergillus versicolor, A. sydowi, A. nidulans, Bipolaris spp., Chaetomium udagawae and C. thielavioideum, and Emericella spp. These molds are capable of producing ST in relatively large amounts on a given substrate. For example, on corn meal, up to 0.75 to 1.2 g of mycotoxin may be produced by these molds (Holzapfel et al., 1966). A. versicolor can produce as much as 1.3 g ST per 100 g of dried mycelia (Davies et al., 1960). The ability of these molds to produce larger amounts of ST may pose a greater degree of hazard from ST toxicosis, compared to that of aflatoxin, even though ST is a much weaker carcinogen than the latter (Holzapfel et al., 1966). ST has been found in moldy grain, green coffee beans, and cheese, although information on its occurrence in foods is limited. However, it appears to occur much less

frequently than the aflatoxins. In addition, analytical methods for its determination are less sensitive, and it is quite likely that small concentrations in food commodities may not always be detected. Since ST was found to be a contaminant in brown rice stored in warehouses under natural conditions (Manabe and Tsuruta, 1975), several reports have cited the presence of ST in cereals and green coffee beans. The U.S. FDA did not detect ST in an analysis of more than 500 samples in 1974–1975 (Stoloff, 1976). Vesonder and Horn (1985) detected 7.75 µg/g of ST in dairy cattle feed associated with acute clinical symptoms of bloody diarrhea and death. ST is an intermediate in the biosynthesis of aflatoxins and is much like aflatoxins in its chemical structure and biological activity. However, it is not very toxic when compared to AFB1. ST was the first naturally occurring compound identified to contain the furfuran ring system (Hamasaki and Hatsuda, 1977). A bishydrofuran ring fused to a substituted anthraquinone characterizes it. The terminal bond of ST, which is important for its mutagenic and carcinogenic activity, is similar to AFB1. Its chemical structure and those of some related compounds are shown in Figure 11.15. The most economically important member of this group is ST from A. versicolor. Other species that produce this toxin include aspertoxin (3-hydroxy-6,7-dimethoxydifuroxanthone) (Rodricks et al., 1968a, 1968b; Waiss et al., 1968), o-methylsterigmatocystin (Burkhardt and Forgacs,

1968), and dihydro-o-methylsterigmatocystin (Cole and Kirksey, 1970) from A. flavus; and 5-methoxysterigmatocystin (Holker and Kagal, 1968), 6-demethylsterigmatocystin (Elsworthy et al., 1970), dihydrosterigmatocystin (Hatsuda et al., 1972), and dihydrodemethylsterigmatocystin (Hatsuda et al., 1972) from A. versicolor. The major differences among the various sterigmatocystins are the presence or absence of unsaturation in the difurano ring system (similar to AFB1 and AFB2) and the substitution pattern on positions 6, 7, and 10 of the xanthone ring system and/or position 3 of the difurano system. Similarity in structure of ST and AFB1 suggests that these mycotoxins have a common biogenetic pathway or that the aflatoxins may be derived from ST and/or vesicolorin-type precursors (Holker and Underwood, 1964; Holker and Mulheirn, 1968; Rodricks, 1969; Biollaz et al., 1970). A. parasiticus can convert ST to AFB1 (Singh and Hsieh, 1976). The physical properties of ST are summarized in Table 11.10 together with those of several other mycotoxins described in this chapter. This particular mycotoxin crystallizes as pale yellow needles and is readily soluble in methanol, ethanol, acetonitrile, benzene, and chloroform. It reacts with hot ethanolic KOH and is methylated by methyl sulfate and methyl iodide. Methanol or ethanol in acid produces dihydroethoxysterigmatocystin. Although ST is primarily a hepatotoxic agent, its hepatotoxicity and tumorigenic potency are considerably

R1

O

OR2

R4 O O

Figure 11.15

O

OR3

R1

R2

R3

R4

H

H

CH3

H

Sterigmatocystin

H

CH3

CH3

H

O-Methylsterigmatocystin

OCH3

H

CH3

H

5-Methoxysterigmatocystin

H

H

H

H

Demethylsterigmatocystin

H

CH3

CH3

OH

Aspertoxin

Chemical structures of sterigmatocystin and related mycotoxins.


Table 11.10

Physical Properties of Selected Mycotoxins

Mycotoxin

Molecular formula

Molecular weight

Melting point, °C

Sterigmatocystin Cyclopiazonic acid Ochratoxin A

C18H12O6 C20H20N2O3 C20H18ClNO6

324 336 403

246 246 169

Citrinin

C13H14O5

250

179

Patulin Deoxynivalenol T-2 toxin Diacetoxyscirpenol Fumonisin B1 Fumonisin B2 Zearalenone

C7H6O4 C15H20O6 C24H34O9 C19H26O7 C34H59NO15 C34H59NO14 C18H22O5

154 296 466 366 721 705 318

111 131–135 150–151 162–164 Powder Powder 164

Moniliformin Tenuazonic acid

C4HO3Na C10H15NO3

120 197

Oil

Altenuene

C15H16O6

292

190–191

Alternariol Alternariol monomethy ether

C14H10O5 C15H12O5

258 272

350 267

less than those of AFB1. For example, the oral LD50 in mice exceeds 800 mg/kg. The 10-day LD50 in Wistar rats is 166 mg/kg in males, 120 mg/kg in females, and 60 to 65 mg/kg for intraperitoneal administration in males. The intraperitoneal 10-day LD50 for vervet monkeys is 32 mg/kg (Ueno and Ueno, 1978; Van der Watt, 1974). Its hepatocarcinogenic activity is approximately one tenth that of AFB1 (Van der Watt, 1974). As with aflatoxins, mice are less susceptible to ST than are rats. Chronic symptoms of ST poisoning include induction of hepatomas in rats, pulmonary tumors in mice, and renal lesions and alterations in the liver and kidneys of African green monkeys. Rats fed 5 to 10 mg/kg ST for 2 years showed a 90% incidence of liver tumors (Ohtsubo et al., 1978). Toxic effects of ST-fed laboratory animals have included kidney and liver damage and diarrhea (Ciegler and Vesonder, 1983). Cattle that had bloody diarrhea and loss of milk production and in some cases died were found to have ingested feed containing A. versicolor and high levels of ST of about 8 mg/kg (Vesonder and Horn, 1985). Several in vitro mutagenicity tests suggest that ST is, like AFB1, a mutagenic agent that covalently binds to


Ultraviolet absorption, nm 15 200 (325) benzene 20 400 (282) methanol 36 800 (213) 6400 (332) ethanol 22 280 (222) 8279 (253) 4710 (310) ethanol 14 600 (275) ethanol Maximum at 218 ethanol Maximum at 187 cyclohexane None Low Low 29 700 (236) 13 910 (274) 6020 (316) ethanol 5000 (218) 12 500 (277) acid methanol 11 500 (240) 14 500 (280) methanol 30 000 (240) 10 000 (278) 6600 (319) ethanol 38 000 (258) ethanol

DNA at approximately 20% to 30% of the level observed with AFB1 (Purchase and Van der Watt, 1968). The toxicity of ST analogs on primary cell culture was greater for the compounds containing the ∆1,2-furobenzofuran ring system than those for the compounds containing a saturated furobenzofuran ring system (Englebrecht and Altenkirk, 1972). These researchers also showed that a carbonyl group unsaturated in the α,β position and an unsaturated bond in the ∆1,2 position are essential to their carcinogenicity. Also, a methoxy group at position 6 enhances, whereas the one at position 7 decreases the toxicity of these compounds. ST does not appear to be teratogenic; however, conclusive data are lacking (Hood and Szczech, 1983) Wannemacher and colleagues (1991) have reviewed the acute toxicity, carcinogenicity, and metabolism of ST in relation to aflatoxins and other hepatotoxic mycotoxins. 11.2.3

Ochratoxins

Ochratoxins (OTs) are metabolites produced by many Aspergillus and Penicillium species. The Aspergillus species

lites of the culture of A. ochraceus. The toxicity of the esters of OT-A is similar to that of OT-A, whereas those of OT-B are not toxic (Ueno, 1987). Mellein and 4-hydroxymellein, which are structurally related to the dihydroisocoumarin moiety of OT-A, have been isolated from A. ochraceus and other strains.

include A. ochraceus, A. sulphureus, A. sclerotinium, A. alliaceus, A. melleus, A. ostianus, and A. petrakii. The Penicillium species include P. purpurescens Sopp, P. commune Thom, P. viridicatum Wes, P. palitans, P. cyclopium Westing, and P. variabile (van Walbeek et al., 1969; Ueno, 1987; van Egmond and Speijers, 1998). The production of ochratoxins depends on environmental conditions such as water activity and temperature. For A. ochraceus high temperatures (12°C–37°C) are necessary, whereas frigophilic penicillia (4°C–31°C), particularly P. viridicatum, also produce ochratoxins in areas with a colder climate. The latter is a common storage fungus in areas such as Canada, Eastern Europe, Denmark, parts of South America, and the United Kingdom.

Occurrence in Foods The frequency and level of OT-A contamination of agricultural commodities are summarized in Table 11.11. OTA often occurs in stored cereals and has been found in other foods, including coffee, beer, dried fruit, wine, cocoa, and nuts. Speijers and van Egmond (1993) have comprehensively reviewed the literature on the worldwide occurrence of ochratoxins. Sensitive and reliable analytical methods now routinely detect ochratoxins at less than 1-µg/kg levels (ppb).

Chemical Characteristics The ochratoxins are a group of seven closely related fungal metabolites (Figure 11.16). OT-A, the 7-carboxy-5chloro-8-hydroxy-3,4-dihydro-3R-methyl isocoumarinamide of L-β-phenylalanine, is the most important and most common. It is also the most toxic of ochratoxins and is produced in highest yield (Neshiem, 1969). OT-B is a less toxic metabolite and lacks the C-5 chlorine; the methyl and ethyl esters of both OT-A and OT-B are minor metabo-

Physical and Chemical Properties OT-A is a colorless crystalline compound, which exhibits blue fluorescence under UV light. It is highly soluble in polar organic solvents, is very slightly soluble in water, and dissolves in aqueous sodium bicarbonate. On acid hydrolysis, it yields phenylalanine and an optically active

OR

O

OH

O

O CH2

CH

NH

O

C

CH3 R'

Figure 11.16

R"

R

R'

R"

H

Cl

H

Ochratoxin A (OT-A)

H

H

H

Ochratoxin B (OT-B)

Et

Cl

H

Ochratoxin C (OT-C) (OT-A ethyl ester)

Me

Cl

H

OT-A methyl ester

Et

H

H

OT-B ethyl ester

Me

H

H

OT-B methyl ester

H

Cl

OH

4-OH OT-A

Chemical structures of ochratoxin A and related fungal metabolites.


Table 11.11

Natural Occurrence of Ochratoxin A (OT-A) in Agricultural Commodities

Commodity

Country

Samples, no.

Contamination, %

Corn Wheat (red spring) Corn Wheat Sorghum Groundnut Wheat (red winter) Wheat (red spring) Animal feeds and tissues Coffee beans Moldy bread Porcine kidney Porcine serum Porcine serum Nuts Beans Corn

United States Canada India India India India United States United States Canada United States Italy Poland Poland Sweden Germany Bulgaria Bulgaria

4 21 24 24 18 291 286 496 267 1 122 388 279 150 24 22

100 30–50 8 12.5 11 1 2.8 1.1 7.1 50 38 16 5 16.7 27.3

OT-A, µg/kg 110–150 20–100 30–50 50–70 50–200 25–35 15–115 50–200 22–360 80 000 ≥1 1–450 ≥2 0.2–8.6 (ng) 25–27 25–35

Source: Compiled from Ueno (1987).

lactone acid, ochratoxin α. OT-A is a moderately stable molecule and survives most food processing to some extent (Scott, 1996). In biological systems, it binds to serum albumin. Metabolism OT-A is metabolized to isocoumarin and hydroxylated metabolites in both in vivo and in vitro systems. The half-life of the toxin is about 55 hours after either oral or intravenous administration in rats, and approximately 56% of the toxin is excreted via urine and feces, both as the free metabolite and hydrolyzed as ochratoxin α 120 hours after dosing (Galtier et al., 1979). Biotransformation pathways for OT-A metabolism are shown in Figure 11.17. It is likely both that OT-A is directly toxic and that other toxicities result from metabolism. In vitro studies, including primary cultures of hepatocytes, have provided evidence that toxicological effects may be linked to biotransformation processes. On biotransformation, OT-A is converted to 4(R)-4-hydroxyochratoxin A (4R), the major metabolite, and 4(S)-4-hydroxy-ochratoxin A (4S), the minor metabolite (Figure 11.17). In structure-activity toxicity studies in vitro in eukaryotic cell systems (HeLa cells) and in rats and mice in vivo using OT-A, its opened lactone ring form and several analogs have shown that the acute toxicity of this mycotoxin is attributable to its isocoumarin moiety. The lac-


tone carboxyl group may also be involved in its toxicity (Neal, 1998). Toxicological Characteristics In acute toxicity studies, LD50 values vary greatly in different species (Table 11.12). Dogs appear to be especially susceptible to OT-A poisoning. Kuiper-Goodman and Scott (1989) have reviewed the subacute and subchronic effects of several feeding trials lasting up to 90 days or more. OT-A is a potent kidney toxin, and both swine and poultry are very sensitive to it (Jacques, 1988). It acts principally on the first part of the proximal tubules in the kidney and induces a defect in the anion transport mechanism on the brush border of the proximal convoluted tubular cells and basolateral membranes, thus leading to the release of membrane-bound enzymes (Endou et al., 1986). However, its dechloro derivative, OT-B, is nontoxic. A nephrotoxic effect has been demonstrated in all mammalian species tested to date (Scudamore, 1998). Dietary concentrations as low as 200 ppb of OT-A over a 4-month period in pigs produce nephropathy that is characterized by degeneration of the proximal tubules, intestinal fibrosis, and hyalinization of some glomeruli (Krogh et al., 1979). OT-A-induced nephropathy of chickens indicates similar signs, with enlarged and pale kidneys (Ellings et al., 1975). The disease has been experimentally produced in several other species (Carlton and Krogh, 1979).

OH

O

HOOC

Ochratoxin α

O H CH3 Cl OCHRATOXIN A COOH CH2

C H

O

HN

OH

O (4R)-4-Hydroxyochratoxin A

C

O OH

O

COOH

O

CH3 CH2

Cl

HN

C H

C

O H CH3 Cl

COOH CH2

C H

HN

O

OH

C

O O H COOH

CH2OH 10-Hydroxyochratoxin A

OH

H

Cl

CH2

C H

HN

O

OH

C

O O H CH3

(4S)-4-Hydroxyochratoxin A

Figure 11.17

Cl

HO

H

Biotransformation pathways for OT-A metabolism in animals.

Although no true cases of mycotoxic nephropathy in humans have been yet reported, it is quite likely that this potent nephrotoxin, which produces renal disease in several species, can also induce renal disorders in humans on exposure. Because of the distinct similarity between the so-called Balkan nephropathy and the ochratoxin-induced porcine renal disease, OT-A has been suggested to be a toxin associated with Balkan endemic nephropathy (Krogh, 1974; van Egmond and Speijers, 1998). This endemic fatal renal disease in humans occurs in certain rural areas of Bulgaria, Romania, and Yugoslavia. OT-A has been found in human blood at levels of about 1–2 µg/kg in about 50% of the samples in various Western European countries, and in mother’s milk at subµg/kg levels (van Egmond and Speijers, 1998). The significance of the detection of OT-A in human body fluids is not yet clear.


Experimentally, OT-A has been found to be a potent inhibitor of protein synthesis, an immunosuppressive agent, a teratogen, a mutagen, and a carcinogen in laboratory animals (Ueno, 1987; Dirheimer, 1996). IARC (1993) has classified OT-A as a 2B carcinogen (possibly carcinogenic to humans). OT-A is both embryotoxic and teratogenic in a variety of species, including mice, rats, hamsters, chicks, and Japanese quail, but not in pigs (Kuiper-Goodman and Scott, 1989; Scudamore, 1998). The central nervous system is one of the most susceptible targets and is affected at the time of early oncogenesis. This mycotoxin must therefore be viewed with suspicion as a potential human teratogen. The presence of OT-A in foodstuffs is clearly undesirable, although few countries seemed to have introduced statutory control by the late 1990s (FAO, 1997). Despite

11.2.4

Table 11.12 Lethal Dose in 50% of Sample Values for Ochratoxin A in Various Species

Fumigatin (Figure 11.18a) was first noted to be a product of A. flavus isolated from Indian soil that was responsible for pH color changes in the culture broth of the strain (Anslow and Raistrick, 1938). It was also produced by A. fumigatus (Anslow and Raistrick, 1938; Pettersson, 1963). Fumigatin is one of a group of toluquinone compounds (Wilson, 1971). It has marked in vitro inhibitory properties against several gram-negative and gram-positive bacteria. The effective range is approximately the same for both types. Not much is known about the animal toxicity of fumigatin. Since it is structurally similar to quinines and hydroquinones, fumigatin is thought to have toxicity similar to that of the quinones.

LD50 values, mg/kg body weighta Species Mouse Rat Rat neonate Dog Pig Chicken

Oral

Intraperitoneal

Intravenous

46–58.3 20–30.3 3.9 0.2 1 3.3

22–40.1 12.6

25.7–33.8 12.7

Source: Compiled from Kuiper-Goodman and Scott (1989) and Scudamore (1998).

the uncertainties, the FAO/WHO Joint Expert Committee on Food Additives has established a provisional tolerable weekly intake (PTWI) level of 112 ng OT-A/kg body weight (WHO, 1991). A working group of Nordic countries proposed a much lower tolerable daily intake of OT-A of 5 ng/kg body weight (NNT, 1991). O

(a) HO

(b)

CH3

Fumigatin

11.2.5

Aspergillic Acid

Aspergillic acid (Figure 11.18b) was first discovered and named by White (1940) and White and Hill (1943). It is

(c)

CH3

N

CH3

N

CH3 H3C

H3CO

N CH3

O

N

O

CH3

O

OH

Aspergillic acid (hydroxy pyrazinone form)

H 3C

O

(e)

CH2OH

O

H3C

Aspergillic acid (hydroxamic acid form)

Fumigatin

(d)

CH3

OH

CH3

OH

O HO

CH3 O Kojic acid

COOH

(f)

O

CH3 OCOCH3

Terreic acid CH3

(g) HOOC

O

Fumagillin (CH

OCOCH3

O CH3

CH)4COO

O

CH3 H3CO

CH2 H 3C

Figure 11.18

H C

C

CH3

O

Chemical structures of miscellaneous mycotoxins produced by various Aspergillus species.


Helvolic acid

the first of a number of closely related pyrazine fungal metabolites reported. It is a major metabolite of certain strains of A. flavus and other Aspergillus species. Aspergillic acid can exist in either the hydroxamic acid form (2hydroxy-pyrazine-1-oxide, Figure 11.18b) or the 1-hydroxy-2-pyrazinone form (Figure 11.18c). Toxicity of aspergillic acid is related to the hydroxamic acid functionality. Little effect on toxicity is observed for differences in the 3- and 6-position side chain substituents. It is acutely toxic to mice at LD50 values of 100–150 mg/kg, intraperitoneally, but has no chronic effects at sublethal dosages; convulsions are the major manifestation of toxicity (White and Hill, 1943). Chelation of physiologically important ions, such as calcium, by aspergillic acid appears to be the likely mechanism of its toxic action (Wilson, 1971, MacDonald, 1973). 11.2.6

Kojic Acid

Kojic acid (Figure 11.18d) is a relatively common metabolite of several species of Aspergillus, including A. flavus and A. parasiticus. Additionally, certain species of Penicillium, such as P. citrinum and P. rubrum, also produce kojic acid as a secondary metabolite. The name of this acid is derived from koji, the mold starter used in Asian food fermentations. Its chemical structure can be defined as 5hydroxy-2-(hydroxymethyl)-4H-pyran-4-one. It is characterized by a γ-pyrone nucleus substituted at positions 2 and 5 with a hydroxymethyl and a hydroxy group, respectively. Kojic acid is classified as a convulsant mycotoxin, but a relatively large quantity is required to produce severe intoxication or death in animals. For example, a group of mice given kojic acid at dosage levels of 250 to 1250 mg/kg exhibited only mild intoxication but complete immobilization lasting for 3 hours without any lethal effects (Wilson, 1966). The LD50 in 17-g mice was 40 mg through intraperitoneal injection (Morton et al., 1945). Natural cases of kojic acid toxicosis either in animals or in humans have not yet been reported. 11.2.7

Terreic Acid

Wilkins and Harris discovered terreic acid, 2,3-epoxy-6hydroxy-toluquinone (Figure 11.18e), produced by A. terreus, in 1942. It has in vitro activity against several bacteria and fungi as well as a protozoan (Trichomonas vaginalis) that infects the human vaginal tract. The intravenous LD50 in mice has been reported to be 70–119 mg/kg (Cole and Cox, 1981).


11.2.8

Helvolic Acid

Previously known as fumigacin, helvolic acid (Figure 11.18f) is a toxic secondary metabolite found in some isolates of A. fumigatus, Cephalosporium caerulens, and Emericellopsis terricola (Cole and Cox, 1981; Dhumal and Salunkhe, 1992). Repeated intraperitoneal injections to 20-g mice caused peritonitis and superficial liver lesions, with minimal effects on kidneys (Wilson, 1971). Helvolic acid is absorbed from the subcutaneous tissues and gastrointestinal tract and is excreted in an active form in the urine and bile. 11.2.9

Fumagillin

Fumagillin (Figure 11.18g), a metabolite of A. fumigatus, possesses antibacteriophage and amebicidal properties against Entamoeba histolytica (McCowen et al., 1951, Wilson, 1971). However, because of its toxic side effects, such as the peeling of the skin from the palms and soles and sensory disturbances in the hands, feet, and ears, fumagillin has not been approved for use in humans. Fumagillin is not a highly toxic substance since orally dosed mice can tolerate doses up to 2000 mg/kg body weight (Dhumal and Salunkhe, 1992). 11.2.10 β-Nitropropionic Acid Many species of the genus Aspergillus, including A. flavus and A. parasiticus, produce β-nitropropionic acid as a secondary metabolite. It is also known as hiptagenic acid or bovinocidin. The LD50 value for mice is about 250 mg/kg; prominent signs of toxicity are rapid respiration followed by apnea, incardination, marked dilatation of subcutaneous and visceral blood vessels, and mottled liver (Wilson, 1971). 11.2.11 Gliotoxin Gliotoxin, an epidithiodioxopiperazine mycotoxin, is a metabolic product of A. fumigatus, A. terreus, A. chevalieri, Trichoderma lignorum, Penicillium obscurum, and P. terlikowskii (Cole and Cox, 1981; Frame and Carlton, 1988). It possesses potent immunosuppressive activity in vitro (Mullbacher and Eichner, 1984). It is acutely toxic to rabbits (LD50 = 45 mg/kg), mice (50 mg/kg), rats (50 to 65 mg/kg) and hamsters, in which the mortality rate is normally more than 50% after the oral administration of doses ranging from 15 to 35 mg/kg body weight (Cole and Cox, 1981). Clinical signs of toxicity are usually nonspecific; they include decreased activity, tachypnea, and prostration

and, in some cases, diarrhea, low blood pressure, hematuria, and congestion of the liver and kidneys. 11.2.12 Fumitremorgins The group of fumitremorgins includes three toxins: fumitremorgins A, B, and C. All of them are produced by A. fumigatus; the first two are also produced by certain strains of A. caespitosus (Cole and Cox, 1981). Fumitremorgin A is a potent neurotropic mycotoxin, which on intravenous injection causes tremors and generalized tonic-clonic convulsions in experimental animals (Nishiyama and Kuga, 1989). A dose as small as 0.1 mg/kg can induce tonic-clonic convulsions in rabbits and also in mice. Similarly, fumitremorigins B and C are tremorgenic in mice (1 mg per mouse, intraperitoneally) and in day-old cockerels (25 mg/kg, orally), respectively (Cole and Cox, 1981). 11.2.13 Maltoryzine The mycotoxin maltoryzine has been isolated from A. oryzae; it has an intraperitoneal LD50 value of 3 mg/kg in mice, in which it causes muscular paralysis and swollen yellow liver. It has been implicated in at least two cases of feed poisoning in cattle (Iizuka and Iida, 1962; Iizuka, 1974).

11.3 MYCOTOXINS OF PENICILLIUM SPECIES Penicillium, like Aspergillus, comprises many toxigenic species. They are as common as Aspergillus molds, and under favorable conditions are found in practically all types of food products. Penicillia have been found in food products implicated in animal mycotoxicoses. For example, moldy corn toxicosis of various forms seems to be associated with Penicillium species, such as P. rubrum, P. cyclopium, P. viridicatum, and P. urticae (Concon, 1988). Some of the important mycotoxins produced by Penicillium spp. are described in the following sections. 11.3.1

Patulin

Patulin was discovered in a long, intensive search for new antibiotics. It was first isolated in crystalline form from P. claviforme and given the name clavicin. Identical materials from other fungal species were isolated by a number of investigators, each of whom assigned a name based on the fungal source. Therefore, the literature contains many synonyms for patulin, including clavacin, expansin, myocin, penicidin, leukopin, and tercinin. Woodward and Singh


(1949) finally suggested the name patulin with elucidation of the correct chemical structure in 1949. Patulin-producing fungi belong principally to the genera Penicillium and Aspergillus (Table 11.13). P. expansum is probably the most common producer of patulin in the environment. The optimal temperature for the growth of this fungus on whole wheat is 10°C and between 20°C and 25°C for the biosynthesis of patulin. In a screening for production of antifungal and antibacterial molecules by 850 strains of fungi, patulin was produced by 58 fungi, including Mucorales and Fungi Imperfecti. Natural substrates that allow production of patulin by Penicillium spp. include malt, barley, rice, wheat straw, grass silage, soil containing root bark, leaves of apple tree, root residues from other fruit trees, and apples. Occurrence in Foods Brian and associates (1956) first identified food products naturally contaminated with patulin by using antimicrobial methods. Patulin was found at concentrations in excess of 1000 ppm in the sap of apples naturally contaminated with P. expansum. Several researchers have reported the natural contamination of apple products with patulin (Drillean and Bohuon, 1973; Eyrich, 1975; Harwig et al., 1973). Thus, contamination of apple juice and other apple products with patulin has occurred when unsound apples were used. The

Table 11.13 Patulin-Producing Fungi and Synonyms for Patulin Fungal species P. expansum Penicillium spp. P. claviforme P. patulum P. melinii P. urticae P. equinum P. novae-zeelanliae P. leucopus P cyclopium, P. griseofulvum P. equinum P. divergens P. lapidosum Aspergillus clavatus A. giganteus A. terreus Byssochylamys nivae Mold species

Name given to substance Expansine, clavacin Penicidin Claviformin, clavatin Patulin, clavatin Clavacin Clavacin Clavacin Clavacin Patulin Patulin Patulin Patulin Patulin Patulin Clavacin, patulin Gigantic acid, claviformin Clavacin Clavacin Myocin c

increased mechanization of apple harvesting has increased the possibility of including unsound apples in processing. The results of a limited survey of cider mills revealed that patulin contamination depended on the proportion of decayed apples used in making fresh apple cider (Wilson and Nouvo, 1973). Frank (1977) reported finding patulin in spontaneously molded pears, peaches, apricots, bananas, pineapples, and grapes. Buchanan and colleagues (1974) also reported detection of patulin in mechanically damaged pears and grapefruit. Patulin has been found in commercially available apple products. Scott and coworkers (1972) found 1 ppm patulin from 1 of 12 commercially available apple cider samples in Canada. Kiermeier (1985) has summarized the analytical data from more than 67 reports during 1980–1983; of 356 samples analyzed, 16 apple juice and related products were contaminated with more than 50 ppb of patulin. Patulin has been reported in spontaneously molded baked goods, including bread, at concentrations of 100 to 300 ppb (Reiss, 1972, 1973). It has also been found in unroasted cocoa beans, grape juice, and commercial tomato paste. Food commodities that have been contaminated with patulin-producing fungi are listed in Table 11.14. The established tolerance level of patulin is zero in all foods in Belgium and 50 ppb in apple juice in Norway, Sweden, and Switzerland (Schuller et al., 1982, 1983). In most countries, patulin is not usually covered by statutory regulation, but quality is sometimes controlled by the setting of a “guideline” or a “recommended” maximal conTable 11.14 Food Commodities Contaminated with PatulinProducing Fungi Wheat flour Refrigerated dough products Cereals and legumes

Pecans Fruits (apricots, crab apples, persimmons, pears, grapes, apples) Fruit juices Meat Poultry feed Cheese, Swiss Cheese, Cheddar Bread

Aspergillus terreus, A. clavatus, P. patulum, P. cyclopium A. terreus, P. urticae Penicillium expansum, P. urticae, A. terreus, A. clavatus, Byssochlamys nivea P. expansum P. expansum, B. nivea

B. nivea P. expansum, P. urticae, P. melinii, P. claviforme P. patulin, P. cyclopium Penicillium spp. Penicillium spp. P. patulum, P. cyclopium


centration, commonly set at 50 ppb in most countries. On the basis of reproduction and long-term carcinogenicity studies in rats and mice, the Joint Expert Committee on Food Additives of the World Health Organization has allocated a provisional tolerable weekly intake of 7 µg patulin/kg body weight (WHO, 1991). Chemical Characteristics Patulin is a β-unsaturated lactone, 4-hydroxy-4H-furo(3,2C)pyran-2(6-OH)one, with an empirical formula of C7H6O4 and a molecular weight of 154 (Figure 11.19a). Its chemical structure was elucidated by Woodward and Singh (1949) and later confirmed by Dauben and Weisenborn (1949). Patulin is optically inactive in spite of having an asymmetrical carbon atom in its structure. It can be isolated as colorless to white crystals from ethereal extracts. It is soluble in water and polar organic solvents. It undergoes all the chemical reactions expected of a secondary alcohol, including esterification to form benzoate, cinnamate, and monoacetate. It reacts as a simple carbonyl compound after ring opening at the hemiacetal function and can form semicarbazone, oxime, and phenylhydrazone. Patulin is generally stable in acidic solutions at pH 2 or below. It is, however, unstable in alkali and is slowly decomposed in distilled water and methanol at ambient temperatures. Patulin is stable in grape and apple juice at 22°C and in dry corn but is unstable in orange juice, flour, baked bread, wet corn, ground sorghum, cheese, or apple juice fermented with Saccharomyces spp. The instability of patulin and its disappearance from various food commodities have been attributed to patulin reactivity with sulfhydrylcontaining amino acids in proteins (Ciegler, 1977). Metabolism Distribution and metabolism studies of patulin are limited, and no metabolic products have yet been identified. It is quite likely that the metabolic fragments or conjugated metabolites of patulin either are bound to the cell membranes or become incorporated into the cellular components. Results of metabolic studies using 14C-patulin show that it is excreted principally via the feces and urine. The major retention and storage site is the erythrocyte (McKinley and Carlton, 1991). Patulin inhibits aerobic respiration in several systems (Singh, 1967; Stott and Bullerman, 1975). The available evidence supports the hypothesis that patulin acts on respiration and that a modified form of patulin may be the real toxic agent. The exact site of patulin action on respiration is not known. It is quite possible that such a site may occur in the respiratory chain before the terminal electron

transport chain of aerobic respiration. Inhibition of anaerobic bacterial growth also supports a prior site of action. Cytological effects of patulin can be interpreted on the basis of respiratory inhibition. Inhibitors of respiration, such as cyanide and dinitrophenol, also inhibit mitosis, but anabolic systems, such as nucleic acid synthesis and protein synthesis, are relatively insensitive to the effects of patulin. Toxicology Patulin possesses wide-spectrum antibiotic properties and has been tested extensively in human subjects to evaluate its ability to treat the common cold. However, its effectiveness has never been proved and its use to treat medical conditions has not been pursued because it irritates the stomach, causing nausea and vomiting. No natural outbreak of disease in animals or humans has been definitely attributed to patulin contamination. However, in several disease outbreaks in cattle, known patulin-producing fungi were isolated from suspect feedstuffs. The LD50 values of patulin have been determined in various animal species (McKinley and Carlton, 1991). The ranges of LD50 values by routes of administration were oral, 25 to 46; subcutaneous, 10 to 33; intraperitoneal, 5 to 15; and intravenous, 15 to 25 mg/kg body weight. Clinical signs after patulin administration include restlessness, increased activity, and dyspnea within 5 minutes of subcutaneous administration; and locomotor difficulties, abdominal pain, and dyspnea after oral, intraperitoneal, and intravenous administration. Convulsions preceding death were reported after intravenous administration. In acute and short-term studies patulin causes gastrointestinal hyperemia, distention hemorrhage, and ulceration. Patulin is also toxic to the rabbit, guinea pig, dog, nonhuman primate, cat, chicken, pigeon, sheep, frog, and fish. It is also toxic to a wide variety of microorganisms and cell types in vitro. Patulin injected in large amounts over a 2-month period was carcinogenic, resulting in induction of sarcomas at the injection site (Dickens and Jones, 1961). In longterm studies at lower dose levels, these effects were not observed. Patulin has been shown to be immunotoxic and neurotoxic. The International Agency for Research on Cancer (IARC, 1986) concluded that no evaluation could be made of the carcinogenicity of patulin to humans and that there was inadequate evidence in experimental animals. Similarly, patulin has not yet been found to be teratogenic in mammals, although it can be fetotoxic and is teratogenic to the chick. Since it is both a highly toxic inhibitor of RNA polymerase and possibly carcinogenic as well, patulin exposure should obviously be avoided


regardless of whether or not it is a likely teratogen in humans. 11.3.2

Citreoviridin

The mycotoxin citreoviridin (Figure 11.19b) was first isolated from P. citreoviride growing on rice and later from many other species of Penicillium (Hirata, 1947; Ueno, 1985). Yellowed rice infected by fungi is the major source of this mycotoxin. Citreoviridin is a neurotoxin that causes paralysis in the extremities of laboratory animals, followed by convulsions and respiratory arrest (Ueno and Ueno, 1972). The LD50 values of citreoviridin in male mice were reported to be 11 (subcutaneous), 7.5 (intraperitoneal), and 29 (oral) mg/kg body weight. Acute signs of toxicity include early onset of progressive paralysis in the hindlegs and flank followed by vomiting, convulsions, and respiratory arrest. Similar signs of poisoning are seen in other species, including cats and dogs. Some of these toxic manifestations may be attributed to inhibition of mitochondrial ATPase activity by citreoviridin (Gause et al., 1981). 11.3.3

Penicillic Acid

Penicillic acid (Figure 11.19c) was first isolated from a mold culture in 1913 as part of a study of corn deterioration (Alsberg and Black, 1913). It is produced by a large number of Penicillium species and also by some species of Aspergillus (Cole and Cox, 1981). Toxigenic strains of these fungi produce substantial amounts of penicillic acid in corn, rice, barley, sorghum, and oats. It also occurs naturally in Swiss cheese (Bullerman, 1976). Although it has significant activity against many gram-negative and a few gram-positive bacteria, its potential as an antibiotic diminished when it was found to be too toxic in clinical use (Oxford, 1942). The LD50 of penicillic acid in mice by oral, intravenous, and subcutaneous routes was found to be 600, 250, and 110 mg/kg, respectively (Murnaghan, 1946). Major manifestations of toxicity include CNS signs and liver lesions. The mechanism of toxicity of penicillic acid may involve the inhibition of sulfhydryl group–containing enzymes. It also shows synergism with other mycotoxins in exhibiting and enhancing their toxic effects. The presence of penicillic acid as a contaminant in foods and feeds is of concern because of its carcinogenic properties (Dickens and Jones, 1963b). In contrast to its carcinogenic potential, penicillic acid also seems to possess antitumor activity (Suzuki et al., 1971).

O

(b) Citreoviridin

OCH3

O

CH3

CH3

HO OH

O

O

OH

O

(a) Patulin

CH3

OCH3

OCH3

O

HOOC

HO C

CH3

OH

H3CO

H3C

O CH3

O

O O

O

H3CO

O

O CH3

Cl

CH2

CH3

(d) Griseofulvin

(c) Penicillic acid

OH

OH

O

CH3 CH3

(e) Citrinin

OH

O

H3C

H3C

O

OH

(f) Luteoskyrin OH

CH2OH O H N

O C2H5 N H HN

O

OH

O

O

OH

H N

COOH O

O O

OCH3

N CH3

(g) Islanditoxin Cl


CH3

Cl

(h) Mycophenolic acid

CH3 O N

H3C

OHO

CH3 O

HO

CH3

OH

O

NH

(i) Decumbin

H3C(CH2)5

(j) Cyclopiazonic acid

OH OH

O O

O

H3C

OCH3 OH

O

O

OH

O

O O O

O

HO

OH

O H3CO

CH3

OH O

(k) Rubratoxin A

(m) Viomellein

O

H3C(CH2)5

OH OH O

O

O OCH3

H3C

OH O

O

O

O

O

O

O O O

OH

O H3CO

OH O

(l) Rubratoxin B

(n) Xanthomegnin

O

Figure 11.19

CH3 O

Chemical structures of mycotoxins produced by Penicillium species.


11.3.4

Griseofulvin

Griseofulvin (Figure 11.19d) is a metabolic product of P. griseofulvum and several other species of Penicillium. It is used as a systemic therapeutic agent for cutaneous fungal infections. In spite of the clinical effectiveness of griseofulvin and the ability of humans and animals to tolerate relatively large doses given daily for several weeks, the serious nature of toxic response observed has restricted its therapeutic use somewhat. Toxic symptoms of griseofulvin poisoning include angioneurotic edema, erythema, urticaria, vesicular and macular eruptions, and photosensitivity of the skin. Other acute toxicity signs include nausea, vomiting, diarrhea, vertigo, blurred vision, headache, transient leukopenia, granulocytopenia, punctate basophilia, and monocytosis (Dhumal and Salunkhe, 1992). Griseofulvin is carcinogenic and has been found teratogenic in rats and cats (Scott et al., 1975). 11.3.5

Citrinin

Citrinin (Figure 11.19e) was first isolated from P. citrinum by Hetherington and Raistrick (1931). It is produced by many species of Penicillium and Aspergillus. The most important fungus is P. citrinum, which was reported as one of the causative fungi of “yellow rice toxicosis” in Japan (Saito et al., 1971). Other Penicillium species that produce citrinin include P. fellutanum, P. lividum, P. implicatum, P. jeneni, P. citreoviride, P. steckni, P. expansum, P. velutinum, P. canescens, P. notatum, P. viridicatum, P. palitans, and P. claviforme. The aspergilli that produce this mycotoxin include A. terreus, A. niveus, A. candidus, Clavariopsis aquata, and Blennoria spp. Citrinin is (3R-trans)-4,6-dihydro-8-hydroxy-3,4,5trimethyl-6-oxo-3H-2-benzopyran-7-carboxylic acid (Figure 11.19e). TLC and HPLC can isolate it. Nuclear magnetic resonance has been employed to confirm its structure in the presence of other mycotoxins. Citrinin crystallizes as lemon-colored needles, melting at 172°C. It is sparingly soluble in water but soluble in dilute sodium hydroxide, sodium carbonate, or sodium acetate; in methanol, acetonitrile, and ethanol; and in most other polar organic solvents. It is likely to be degraded by heat and alkali. Citrinin has been found in Indian groundnuts infected with A. flavus, P. citrinum, and A. terreus (Subramanyam and Rao, 1974). Citrinin-producing strains of Penicillium spp. also have been found as contaminants of sausage (Ciegler et al., 1972). Citrinin often occurs in conjunction with ochratoxin A, another mycotoxin capable of altering renal function. The mycotoxin is found mainly in rice and other cereals, such as barley, wheat, rye, and oats.


Citrinin is rapidly absorbed irrespective of the route of administration and localized in the liver and kidneys. Under normal conditions, citrinin and/or its metabolites possibly undergo enterohepatic circulation with ultimate disposition through the urine, with the liver as the primary organ of metabolism (Reddy and Berndt, 1991). Similarly to OT-A, citrinin is a potent nephrotoxin. A renal disease, porcine nephropathy, was associated with the feeding of moldy cereals to farm animals (Larsen, 1928). This disorder of the kidney function was later reproduced by feeding contaminated barley and rye to pigs (Nielsen and Hesselager, 1965) and by feeding purified citrinin (Krogh et al., 1973). The nephropathy is characterized by degeneration of the tubules, with subsequent cortical fibrosis along with functional impairment of tubular activity. Similar nephrotoxic action of citrinin is observed in guinea pigs, turkeys, ducklings, beagle dogs, and swine. The mouse is relatively resistant, whereas the pig is relatively sensitive to the nephrotoxic effects of citrinin. The LD50 of citrinin was found to be about 50 mg/kg for oral administration to rat, 35 to 58 mg/kg (intraperitoneally) to the mouse, and 19 mg/kg (intraperitoneally) to the rabbit. Scott (1977) has reviewed the toxicologic characteristics of citrinin. Citrinin alone is not tumorigenic in rats, except that it synergistically increases the renal tumor induced by a nephrotoxic chemical, N-(3,5-dichlorophenyl)succinimide (DDPS) (Shinohara et al., 1976). Citrinin is an embryocidal, fetotoxic, and mildly teratogenic compound (Reddy and Berndt, 1991). Mice exposed to citrinin (30 or 40 mg/kg) on gestation day 6, 7, 8, or 9 experienced reduced fetal weight gain, increased fetal lethality, and increased maternal lethality rates. The combination of citrinin with OT-A increased the occurrence of gross, soft tissue, and skeletal malformations (Mayura et al., 1984). Little appears to be known of citrinin’s mechanism of action, however, and much more extensive testing would be required to establish its teratogenicity or its lack thereof in mammalian species. 11.3.6

Luteoskyrin and Islanditoxin

Luteoskyrin, a yellow anthraquinonelike pigment synthesized by P. islandicum Sopp, contaminates rice, maize, and other cereals, especially in Eastern countries, where human malignant and nonmalignant hepatomas are more frequent. P. islandicum was first isolated in 1912 from skyr, a kind of yogurt produced in Iceland, and the pigments isolated from this fungus were named after the domestic name. Luteoskyrin, a yellow lipophilic toxin, is a substituted bis-polyhydroxy-dihydro-anthraquinone (Figure

11.19f). Islanditoxin is a hydrophilic toxic cyclic peptide (Figure 11.19g). The toxicity of luteoskyrin varies according to the dose and diet, strain, sex, and age of the animal species. For a 10-g mouse, the LD50 is 1.5 mg, subcutaneously, and 2.2 mg, orally. It is known to be toxic to mice and rats, inducing chronic liver injuries, such as malignant and nonmalignant hepatomas (Ueno et al., 1971; Dhumal et al., 1991). Liver cirrhosis has been noted in rats fed a cereal diet contaminated with the fungus. This toxin is also cytotoxic to HeLa cells and to rat liver, kidney, and lung cells over a concentration range of 0.32 to 1.0 µg/ml (Umeda et al., 1972). Luteoskyrin is lipophilic and relatively slow-acting, and the liver damage caused by it is characterized by centrilobular necrosis and diffuse fatty metamorphosis of the liver cells. The mitochondria that control energy production and the nuclei as the information center are both affected by luteoskyrin. It also binds to DNA, causes pigment damage, and alters the activity of DNA-dependent RNA polymerase (Dhumal et al., 1991). Luteoskyrin, therefore, is a potent cytotoxic and mutagenic agent. Islanditoxin is also extremely hepatotoxic, causing severe liver damage, hemorrhaging, and death. LD50 values for 10-g mice range from 4.75 µg, subcutaneously, to 65.5 µg, orally. It interferes with carbohydrate metabolism by causing the disappearance of glycogen granules in the injured liver. 11.3.7

Mycophenolic Acid

Mycophenolic acid is produced by many species of Penicillium, among which P. brevicompactum and P. stoloniferum appear to be most common (Cole and Cox, 1981). It contains a five-membered lactone ring fused to a benzene moiety (Figure 11.19h). Mycophenolic acid is a relatively less toxic fungal metabolite. Its oral and intravenous LD50 values for mice have been reported to be 2.5 g/kg and 550 mg/kg, respectively (Carter et al., 1969). The compound exhibits antibacterial, antifungal, antiviral, and antitumor activity (Wilson, 1971; Cole and Cox, 1981). In food, it has so far been reported only in blue cheese and starter cultures of P. roqueforti (Lafont et al., 1979). 11.3.8

Decumbin

Decumbin (Figure 11.19i) is a toxic metabolite of P. decumbens, isolated by Singleton and coworkers (1958) from corn spoiled in storage. The compounds, brefeldin isolated from the culture filtrate of P. brefeldianum (Harri et al., 1963) and cyanein obtained from that of P. cyaneum


(Betina et al., 1965) and P. simplicissimum (Betina et al., 1966), were later found to be identical to decumbin. Administration of decumbin at levels of 250, 300, and 400 mg/kg to fasted rats resulted in 0%, 80%, and 97% mortality rates, respectively. The acute signs of toxicity began with a rapid onset of anorexia followed by diarrhea, lethargy, labored breathing, cyanosis, stupor, and subsequent death in about 24 hours (Betina et al., 1966). Decumbin is an inhibitor of the synthesis of nucleic acids and proteins in microbial and animal cells. 11.3.9

Cyclopiazonic Acid

Cyclopiazonic acid, a toxic indole tetramic acid (Figure 11.19j), is a toxic metabolite of P. cyclopium and P. puberulum. It can also be produced by certain strains of A. flavus (Yates et al., 1987). It has been detected in naturally contaminated mixed feeds, corn, groundnuts, cheese, and other foods and feeds. Cyclopiazonic acid only appears to be toxic when present in high concentrations. It is toxic to rats; the oral and intraperitoneal LD50 values are 36 and 2.3 mg/kg body weight, respectively (Ohmomo et al., 1973). It is also toxic to mice, chickens, rabbits, dogs, and pigs (Yates et al., 1987). The clinical signs of toxicity include inactivity, diarrhea, anorexia, rough hair coats, tremors, and death. Histopathological examinations have shown degenerative changes and necrosis in the liver, spleen, kidney, pancreas, salivary glands, and muscles. Its effects on the hepatic endoplasmic reticulum and HeLa cells suggest that it is an inhibitor of protein synthesis (Hinton et al., 1985; Zaera et al., 1983). Cyclopiazonic acid can cooccur with aflatoxins (Takashi et al., 1992) and may enhance the overall toxic effect when it does (Cole, 1986; Scudamore, 1998). The lack of authentic human exposure data precludes an assessment of possible health effects. However, “Kodua” poisoning in India resulting from ingestion of contaminated millet seeds has been linked to this toxin. It has pharmacological properties similar to those of the antipsychotic drugs chlorpromazine and reserpine in mice and rabbits. Near-lethal doses of 11 to 14 mg/kg body weight induce continuous involuntary tremors and convulsions. It may be able to produce similar neurotoxic effects in humans. 11.3.10 Rubratoxins Burnside and associates (1957) reported a disease in pigs and cattle caused by the consumption of fungally infected corn. Of 13 cultures of fungi isolated from such toxic corn,

only 2 were shown to cause illness and death when fed to experimental animals. One of the cultures was found to be P. rubrum, which produced a mycotoxin. Townsend and colleagues (1966) first described the rubratoxins as pure compounds, isolated from crude preparations of P. rubrum culture filtrate. The compounds were named rubratoxin A and B on the basis of the difficulty of isolation of the toxic fractions; the former was more easily isolated. The major and most toxic constituent isolated was rubratoxin B (Moss et al., 1967). Rubratoxin A (Figure 11.19k) is a dihydro-derivative with one of the anhydride groups reduced to lactol. Rubratoxin B is a cyclic bisanhydride (Figure 11.19l). The rubratoxins are unusual compounds in that they possess relatively stable anhydride groups. The aldehyde lactol group in rubratoxin A is also an unusual structural feature (Moss, 1971). Rubratoxin B is the major metabolite, and the head-to-tail, head-to-tail coupling of two C13 units produces it. Penicillium rubrum Stoll has been isolated from a variety of cereal and legume products, corn, bran, sunflower seeds, and peanut pods (Scott, 1965). However, the role of these feeds in natural outbreaks of mycotoxic disease remains undetermined (Newberne, 1974; Engelhardt and Carlton, 1991). Both the rat and the mouse excrete rubratoxin B or its metabolite(s) fairly rapidly after initial accumulation in the liver and kidneys. This probably accounts for the development of lesions in these two organs. The long elimination half-life for rubratoxin B in the plasma suggested that enterohepatic circulation was taking place (Unger and Hayes, 1979). The absence of detectable amounts of glucuronide or sulfate conjugates in the plasma and urine indicated that most of the conjugated rubratoxin produced by the liver was being excreted in the bile. Rubratoxin B interferes with several hepatocellular biochemical mechanisms. Decreases in hepatic ATP content (Hayes et al., 1978), ATPase activity (Unger et al., 1978), and hepatic-reduced glutathione content (Watson and Hayes, 1982) could all contribute to hepatocellular degeneration and necrosis. Rubratoxin B also has a strong affinity for sulfhydryl groups (Phillips and Hayes, 1979). Inactivation of membrane or enzymic sulfhydryl groups due to binding of the toxin could initiate membrane damage, leading to cellular degeneration and necrosis. The differences in target organ, lesion distribution, and lesion severity in the animal species evaluated may be related to differences in metabolism among the various species and/or in the biochemical susceptibility of cell populations in the various organs of the different species to the toxic effects of rubratoxin B.


The LD50 values for rubratoxin A and B are 6.6 and 3.0 mg/kg, respectively. Rubratoxin B has been shown to be mutagenic as well as teratogenic (Hood et al., 1973; Evans et al., 1975). 11.3.11 Viomellein and Xanthomegnin Several fungi, such as P. viridicatum and A. ochraceus, produce the mycotoxins viomellein and xanthomegnin (Figure 11.19m and 11.19n). In mice fed these mycotoxins for 10 days identical lesions develop, predominantly in the liver. These include necrotizing cholangitis, focal hepatic necrosis, and hyperplasia of the biliary epithelium, with only minor changes in the kidneys (Carlton et al. 1976; Ueno, 1987). Hald and coworkers (1983) surveyed these mycotoxins in a barley batch associated with field cases of mycotoxic porcine nephropathy. The data revealed the presence of 1 ppm viomellein, along with 1.9 mg OT-A and 0.8 ppm citrinin. These findings indicate the natural cooccurrence of these nephrotoxic and hepatotoxic mycotoxins in barley. Systematic surveys of the natural occurrence of these mycotoxins in foods are required for their evaluation in food toxicology.

11.4 MYCOTOXINS OF FUSARIUM SPECIES 11.4.1

Zearalenone

Zearalenone, also known as F-2 toxin, is produced by several species of Fusarium. These include F. tricinctum, F. gibbosum, F. roseum, and three subspecies of the latter, F. roseum culmorum, F. roseum equisetti, and F. roseum graminearum (Gibberella zeae in sexual stage) (Urry et al., 1966; Concon, 1988; Dhumal and Salunkhe, 1992). Under favorable growth conditions, these Fusarium species have been found to produce this mycotoxin in corn, barley, mixed feeds, wheat, oats, and sorghum and have caused toxicosis in livestock in various countries. Generally, zearalenone concentrations are well below 1 ppm in processed cereal foods (Morehouse, 1985). In the United States and Canada, contamination of grains by zearalenone is frequently encountered. In these countries, the mycotoxin is most commonly found in cornbased products such as breakfast cereals and cornmeal. In Japan, high concentrations of this mycotoxin sometimes occur as a cocontaminant with deoxynivalenol and nivalenol (Jelinek, 1987). However, no cases of F-2 toxicosis in humans have been reported. Zearalenone is only partly decomposed by heat. Approximately 60% remains unchanged in bread, and about 50% survives the production of noodles (Matsuura and

Yoshizawa, 1981). In dry milling of corn, concentrations in the main food-processing fractions including flour and grits are reduced by 80% to 90%, although increased concentrations are found in bran and germ (Bennett et al., 1976). Zearalenone is a phenolic resorcyclic acid lactone (Figure 11.20a). In fungal cultures, a number of closely related metabolites are also formed. However, there is only limited evidence that these occur in foodstuffs. There is experimental evidence for some transmission of zearalenone and α- (Figure 11.20b) and β-zearalenols (Figure 11.20c) into the milk of sheep, cows, and pigs fed high concentrations (Mirocha et al., 1981). Zearalenone is a white crystalline compound that exhibits blue-green fluorescence when excited by longwavelength UV light (360 nm) and a more intense green fluorescence when excited with short-wavelength UV light (260 nm). It is slightly soluble in hexane and progressively more so in benzene, acetonitrile, methylene chloride, methanol, ethanol, and acetone. It is also soluble in aqueous alkali. Zearalenone and its derivatives produce estrogenic effects in farm animals (pigs and cattle) as well as in laboratory animal species. Its uterotropic activity is exhibited in a similar manner to that of β-estradiol. It binds with the cytosolic estrogen receptor of uterine tissue, followed by translocation to a nuclear receptor and induction of messenger RNA (mRNA) synthesis (Kawabata et al., 1982). Metabolic studies have shown that the hepatic zearalenone-reductases catalyze the transformation of zearale-

none into α-and β-zearalenols. The estrogenic activity of α-zearalenol is much higher than that of the parent compound (Ueno and Tashiro, 1981). Pigs appear to be the species most affected by the consumption of feed contaminated with zearalenone. The estrogenic syndrome in pigs is characterized by swollen, edematous vulva and enlarged mammary glands in females; shrunken testes in young males; a dramatic increase in the weight of the uterine horn of gilts; and possible abortion in pregnant sows and gilts. In sows consuming feed with 25 ppm zearalenone infertility, constant estrus, pseudopregnancy, small litter sizes and birth weights, and fetal malformations can develop (Chang et al., 1979). Although swine have been found to be the most sensitive domesticated animal to zearalenone, calves have been reported to show earlier sexual maturity; dairy cows have been reported to have vaginitis, prolonged estrus, and infertility (Palti, 1978); and sheep are reported to become sterile (Towers and Sprosen, 1992). The effective dose for sheep may be approximately 1 ppm. Poultry appear to be more resistant to the effects of zearalenone than are swine. The acute toxicity of zearalenone is quite low. The LD50 values (milligrams per kilogram body weight) for oral and intraperitoneal administration are, respectively, >2000 and >500 in the mouse, >4000–10,000 and >5500 in the rat, and >15,000 for oral administration in chickens (Scudamore, 1998). Subacute and subchronic toxicity studies of up to 14-week duration indicated that most effects were due to the estrogenicity of zearalenone (KuiperGoodman et al., 1987). In mice, it caused atrophy of semi-

(b) α-Zearalenol

(a) Zearalenone OH

O

OH

CH3

HO

O

HO

OH

O O

CH3 O

O

OH

O

O

CH3 O

HO

OH (c) β-Zearalenol

Figure 11.20

Chemical structures of mycotoxins produced by Fusarium species.


H

O Na (d) Moniliformin

nal vesicles and testes, squamous metaplasia of the prostate gland, osteoporosis, myelofibrosis of bone marrow, cytoplasmic vacuolization of the adrenal gland, hyperkeratosis of the vagina, and endometrial hyperplasia. Makela and associates (1995) compared the estrogenic potency of zearalenone with that of other plant-derived estrogens in MCF-7 or T-47D breast cancer cells and concluded that, in comparison with 17-β-estradiol, it is one of the most potent natural xenoestrogens. Zearalenone has a potential for prenatal toxicity and possibly for teratogenicity. Evidence for genotoxicity has been contradictory, but Pfohl-Leszkowicz and coworkers (1995) showed it to be genotoxic in mice. The International Agency for Research on Cancer (IARC, 1993) concluded that there was limited evidence in experimental animals for the carcinogenicity of zearalenone. Because of its anabolic action on promoting growth hormone and insulin levels, zearalenol, a derivative of zearalenone, has been extensively used as a growth promoter in ruminants (Knight, 1981). Probably because of its low acute toxicity, so far there are no reports of adverse effects of zearalenone and its derivatives in humans. Nevertheless, because chronic low doses have adverse biological effects as a result of their potent anabolic properties and their possible presence in milk, in edible tissues, and in products like beer, zearalenone may cause human health problems after long-term exposure (Ueno, 1985; WHO, 1979; Scudamore, 1998). 11.4.2

Moniliformin

Moniliformin, a sodium or potassium salt of 1-hydroxycyclobut-1-ene-3,4-dione (Figure 11.20d), has been isolated from F. moniliforme, a fungus that causes southern leaf blight in the United States, and also from F. fusarioides (Cole et al., 1973; Rabie et al., 1978). Data on the occurrence of moniliformin in food are scarce. Thiel and colleagues (1982) showed that levels up to 12 mg/kg occurred in corn intended for human consumption in the Transkei. More recently analysis of imported corn-milled products destined for incorporation into animal feeding stuffs in the United Kingdom showed that 60% of the samples were contaminated with concentrations up to 4.6 mg/kg (Scudamore, 1998). Moniliformin has also been shown to occur in other cereals, such as rice and wheat. There is only limited information on its degradation during processing. Moniliformin is a potent inhibitor of mitochondrial pyruvate and α-ketoglutarate oxidation. The reported oral LD50 value for moniliformin in day-old cockerels is 4.0 mg/kg (Cole and Cox, 1981). The intraperitoneal LD50 for female and male mice was, respectively, 20.9 and 29.1


mg/kg. In contrast, the oral LD50 values for female and male rats were 41.6 and 50.0 mg/kg, respectively (Ueno, 1985). The poisoned cockerels showed ascites with edema of the mesenteries and small hemorrhages in the gizzard and intestines. The clinical signs in rats were characterized by a rapid progressive muscular weakness, respiratory distress, terminal coma, and death. Moniliformin does not seem to have any teratogenic effects in experimental laboratory animals (Hood and Szczech, 1983). 11.4.3

Trichothecenes

The ability of fungi to produce a large number of secondary metabolites is striking in the case of the trichothecenes. Several families of imperfect, saprophytic, and plant pathogenic fungi, such as Fusarium, Trichothecium, Myrothecium, Cephalosporium, Stachybotrys, Trichoderma, Cylindrocarpon, and Verticimonosporium spp., produce trichothecenes (Ueno, 1983). More than 80 trichothecenes and trichothecene metabolites have been identified. Furthermore, several interesting trichothecenes have been detected in the biotransformed products catalyzed by animal and microbial systems. In the United States, serious outbreaks of toxicosis among farm animals fed fungally infested corn have been reported. The most toxic fungus frequently isolated from the fungally infected corn in Wisconsin was F. tricinctum, out of which a much more toxic strain, T-2, was selected to produce the toxin. Hence, the toxin was named T-2 toxin. This has become a common name for this trichothecene (Bamburg et al., 1968). The naturally occurring trichothecenes, especially T2 toxin, cause a variety of mycotoxicoses, such as bean hull poisoning in horses in Japan, fungally infested corn toxicosis in various livestock species in the United States, and alimentary toxic aleukia (ATA) of humans and animals in Russia (Joffe, 1986). Interest in trichothecene mycotoxins grew after a widespread outbreak of ATA, or septic angina, in the Soviet Union, which began during World War II and lasted up to 1947. The disease was called septic angina, since it was characterized by a progressive leukopenia and often led to a stage that had some of the signs of sepsis (Joffe, 1986). The disease killed thousands of people and was linked to the ingestion of millet, rye, wheat, and other small grains contaminated with Fusarium spp. The name ATA emphasizes the progressive leukopenia and the characteristics that ingestion of grains (alimentary) and secretion of the toxin by the fungi are necessary for an outbreak of this disease. Deoxynivalenol (vomitoxin) and nivalenol have been associated (together with acetyl deoxynivalenol and T-2 toxin) in an outbreak

of disease in Kashmir, India, in 1987 (Bhat et al., 1989). Some 50,000 people were affected after consumption of bread made from rain-damaged wheat that contained several trichothecenes. Overwintering conditions under low fluctuating temperatures appeared to promote Fusarium sp. mycotoxin production in these grains. The seasonal occurrence of ATA, its endemic nature, and the composition of the affected population suggested the importance of climatic and ecological factors in producing toxins that were found in field grains naturally infected by Fusarium species (Joffe, 1986). The years marked by low temperatures, considerable precipitation, and high humidity were generally associated with outbreaks of the disease in people and animals. Chemical Characteristics Trichothecene mycotoxins have a common sesquiterpene nucleus, which consists of cyclopentane, cyclohexane, and a six-membered oxyrane ring with four methyl groups. The skeleton contains an epoxide ring at C-12,13 and a double bond at C-9,10, characterized as 12,13-epoxytrichothec-9-ene. The numbering system is shown in Figure 11.21. In the early stages of trichothecene research, Ueno and coworkers (1973) classified these mycotoxins into four groups according to their chemical properties and the producing fungi. The types A, B, and C are simple trichothecenes; type D contains macrocyclic trichothecenes. The first (type A) is represented by T-2 toxin, HT-2 toxin, diacetoxyscirpenol, neosolaniol, and others (Figure 11.22). These are produced by F. sporotrichioides, F. sporotrichioides var. tricinctum, and F. poae. Type B trichothecenes are characterized by the presence of a ketone (carbonyl function) group in the C-8 position. This group includes nivalenol, deoxynivalenol, and fusarenon-X and is produced by F. nivale and F. episphaeria (Figure 11.23). The

16

O 1

10

2

11

9

13 6

8 7

O

3

12 5

type C group, represented by crotocin (Figure 11.24) produced by Cephalosporium crotocingigenum, contains a second epoxide at C-7,8 or C-9,10. The type D group includes trichothecenes containing a macrocyclic ring between C-4 and C-5 with two ester linkages. Subsequently, Ueno (1987) added two more groups to this classification. The fifth group (type E) is represented by the macrocyclic trichothecenes in which the macrocyclic ring is opened; the sixth group (type F) is represented by verrucarin K (Figure 11.24), in which 12,13epoxide function is changed to a vinyl linkage, and thereby the oxygen atom in the epoxide ring is removed. These compounds are presumed to be intermediates produced during the biosynthesis of macrocyclic trichothecenes. All natural trichothecenes have the same stereochemical characteristics: α at C-3, C-7, and C-8; and β at C-4 and C-15 for type A and B trichothecenes. The full systematic chemical name of T-2 toxin, therefore, is 3α-hydroxy-4β,15-diacetoxy-8α-(3-methylbutyryloxy)12,13-epoxytrichothec-9-en, and fusarenon-X is 3α-7α15-trihydroxy-4b-acetoxy-12,13-epoxytrichothec-9-en-8one. The chemical structures of various trichothecenes are shown in Figures 11. 22–11.24. The group A trichothecenes are highly soluble in ethyl acetate, acetone, chloroform, methylene chloride, and diethyl ether. The highly hydroxylated type B mycotoxins are relatively polar; they are soluble in methanol, acetonitrile, and ethanol. Physical and Chemical Properties All trichothecenes containing an ester group are hydrolyzed to their respective parent alcohols when treated with alkali. Thus, T-2 toxin and neosolanil are converted to T-2 tetraol and diacetoxy-and monoacetoxy-scirpenol to scirpentriol. Many of the alcohols are unaffected even by hot dilute alkali. Trichothecenes are thus chemically stable and can persist for long periods once formed. Prolonged boiling in water or under highly acidic conditions causes a skeletal rearrangement due to opening of the epoxide ring. Because of the hindered nature of the epoxide and stability of the ring system, reactions of trichothecenes usually proceed in a manner predictable from sound chemical principles. For example, strong oxidizing agents easily oxidize primary and secondary hydroxyl groups to the aldehyde and ketone derivatives (Scudamore, 1998).

4

Occurrence in Food 15 14

Figure 11.21

The numbering system of trichothecene.


Of the 80-odd trichothecenes identified thus far, only a few are detected in cereals and foods. The major naturally occurring mycotoxins of this group are T-2 toxin, diacetoxyscirpenol, deoxynivalenol, nivalenol, and satratoxins. In

H T-2 Toxin

H

H3C O H

HT-2 Toxin

H OH

O

O

CH3

H

H

O OH

O

O

OAc CH2 CH3 OAc

H H3C

H

H3C O H

O

OH H

H H3C

CH3

CH2 CH3 OAc

H

Diacetoxyscirpenol H

H

H3C Neosolaniol

H

H

O

H

H

H

H

H3C T-2 tetraol

H

OAc CH2 CH3 OAc

HO H

CH2 CH3 OAc

H

H

O

CH2 CH3 OH

OAc H

OH

H 3C O H

OH

O

O

H

OH

O

H

HO

H

O

OH

O

H

H

H

H 3C

H OAc

O

OAc H

H H3C

H O

CH3

CH2 CH3 OAc

H

Acetyl T-2 toxin

Figure 11.22

Chemical structures of type A trichothecenes.

H H3C

H

H

H

O

H3C OH

O O

OH

H CH2

CH3

Deoxynivalenol

H

H

H H3C

O

H

O

OAc

H

O

OH

O

OH

O O

OAc

CH2

CH2 CH3

H

OH Fusarenon-X

Chemical structures of type B trichothecenes.


H

OH

H

Figure 11.23

CH3

HO

H

Nivalenol

HO

OH

O

OH

H3C

H O

CH2 HO

H O

HO

CH3 OAc

Diacetylnivalenol

H

H

H H3C

H3C

O O O

O O

H O

H2C

H3C

CH3

O

CH3

H O O

O

Crotocin O

H3C

O Satratoxin H

H3C

OH

OH

H

H

CH3

O O

H O

H2C O

CH3

Verrucarin A

O O H O HO H3C

H O

Figure 11.24

Chemical structures of type C and macrocyclic trichothecenes.

Canada, the United States, South Africa, and England, deoxynivalenol is the sole trichothecene present in corn, wheat, barley, and other cereals (Scott, 1984; Eppley et al., 1984; Marasas et al., 1977). Ueno (1987) reviewed extensively the literature on the contamination of cereal samples from various countries and found both nivalenol and deoxynivalenol in wheat, barley, malt barley, and corn. More than 80% of the samples studied contained both these trichothecenes. Trichothecenes contaminants in cereal flours are readily carried over into foodstuffs such as bread, snack foods, and cake. Residues of trichothecenes, however, are not known to occur in animal products such as meat, milk, and eggs. In contrast, T-2 toxin and diacetoxyscirpenol are found much less frequently. The macrocyclic trichothecenes (types D, E, and F) are rarely found in human food, although their presence in airborne fungal spores may contribute to some forms of sick building syndrome (Croft et al., 1986). Metabolism Metabolic pathways of T-2 toxin in various systems are shown in Figure 11.25. T-2 toxin or its metabolites are primarily excreted from liver via bile into the intestine. It is primarily excreted in feces. In guinea pigs, metabolites of


T-2 toxin include HT-2 toxin, 4-deacetylneosolaniol, 3′hydroxy T-2 triol, 3′-hydroxy HT-2, and several unknowns, after an intramuscular injection (Pace et al., 1985), and T-2 triol, 3′-hydroxy HT-2, T-2 tetraol, the glucuronide conjugate of HT-2, and several unknowns, after a dermal treatment (Kemppainen et al., 1987). Thus, T-2 toxin appears to be metabolized into several hydrophilic compounds, although the trichothecene skeleton itself is not modified by the metabolic action. Toxicological Characteristics The acute toxicity of the trichothecenes varies considerably. T-2 toxin and the macrocyclic mycotoxins are by far more toxic than deoxynivalenol and nivalenol but fortunately are not often found in foods. The symptoms of acute toxicity of trichothecenes appear similar across various species; they include vomiting; inflammation; diarrhea; cellular damage of the bone marrow, thymus, spleen, and mucous membranes of intestine; feed refusal; anemia and leukopenia; and depression of circulating white blood cells. This group of mycotoxins is acutely cytotoxic and strongly immunosuppressive (Scudamore, 1998). The experimental lethal doses, a major characteristic of the severe toxicity of trichothecenes in various species, are summarized in Table 11.15. When given orally or in-

H3C

H3C O

O OH

O

AcO

CH2 CH3

O

OAc AcO

H3C

Neosolaniol

H3C

OH

O

OAc

HO

H3C O

O

OH

O

OAc

O

CH2 CH3

CH3

O

AcO H3C

T-2 toxin

OH

CH2 CH3

CH3

3'-Hydroxy T-2

O OH

O

HT-2 toxin

H3C O

OAc

HO

O OH

O

CH2 HO

CH3

OH

O AcO

15-Deacetylneosolaniol

H3C

H3C

CH3


O O

H3C O

OH OH

HO AcO

CH2 CH3

O OH

O

OH

O

CH2 CH3

HO

H3C

H3C O

CH2 CH3

CH3

O

AcO H3C

H3C

O OH

O HO

H3C O

CH2 CH3

O O

HO

H3C

OH


Metabolic pathways of T-2 toxin in various systems.


OH OH

O

T-2 tetraol

Figure 11.25

CH2 CH3

CH3 3'-Hydroxy HT-2

OH HO

OH

CH2 CH3

CH3 3'-Hydroxy T-2 triol

OH OH

O

T-2 triol

4-Deacetylneosolaniol

O

Table 11.15 Lethal Dose in 50% of Sample Values of the Major Trichothecenes

Trichothecenes T-2 toxin

Diacetoxyscirpenol

Nivalenol Fusarenon-X Deoxynivalenol Crotocin Roridin A Verrucarin A

Verrucarin B Verrucarin J Satratoxin H Satratoxin G a

Animal species

Routeb

LD50, mg/kg

Mouse Rat Swine Mouse Rat Rabbit Swine Mouse Mouse Rat Mouse Mouse Mouse Mouse Rat Rabbit Mouse Mouse Mouse Mouse

i.p. p.o i.v. i.p. p.o i.v. i.v. i.p. p.o. p.o. p.o. p.o. i.p. i.p. i.v. i.v. i.v. i.p. i.p. i.p.

5.2 5.2 1.21 23.0 7.3 1.0 0.37 4.1 4.5 4.4 46 1 000 1.0 1.5 0.87 0.54 7.0 0.5 5.69 1.29

LD50, lethal dose in 50% of sample.

b

i.p., intraperitoneal; p.o., oral; i.v., intravenous. Source: Compiled from Ueno (1985, 1987) and Scudamore (1998).

layed-type hypersensitivity (DTH), antibody formation, and other immunological responses are markedly affected by the trichothecenes. Trichothecenes are therefore classified as immunodepressors (Obara et al., 1984). Alimentary toxic aleukia (ATA) is the most common human trichothecene mycotoxicosis. T-2 toxin is thought to have contributed to the epidemic of ATA in Russia, which was responsible for widespread disease and many deaths. Continuous exposure to trichothecenes results in skin rashes, which may proceed to necrotic lesions. The trichothecenes have not been shown to be mutagenic or carcinogenic but do inhibit DNA and protein synthesis. Although trichothecenes have not been found to be mutagenic in bacterial testing and the IARC has concluded that the carcinogenic potency of these mycotoxins has not been adequately demonstrated, their occurrence in food is still of some concern. This is particularly true because of the immunotoxic effects that these toxins may have. Similarly, the exact mechanism of trichothecene toxicity is not yet clear because the known mechanisms do not explain the severe acute toxicity of these mycotoxins. Further biochemical studies are required at the molecular level, and the exact biochemical entity interacting with trichothecenes needs to be elucidated. This entity may turn out to be a basis for the development of an antidote for trichothecene poisoning. Regulation

traperitoneally, the trichothecenes are acutely toxic at low concentrations. Dosed animals become listless or inactive, and diarrhea and rectal hemorrhaging develop. Necrotic lesions may develop in the mouthparts; the mucosal epithelium of the stomach and small intestine erodes, accompanied by hemorrhage, which may develop into severe gastroenteritis, followed by death (Pier et al., 1980; Scudamore, 1998). The acute toxicity of T-2 toxin suggests that it is rapidly distributed to the target organs (Ueno, 1984). It also appears that all types of cells are susceptible to the toxic effects of trichothecenes; the rapidly proliferating cells, such as epithelial cells and lymphocytes, are more sensitive. Deoxynivalenol, the commonly occurring trichothecene in foods, causes vomiting in pigs at relatively low concentrations. However, pigs are very sensitive to its presence and reject contaminated feed, thereby effectively limiting any further toxic effects. Bone marrow, spleen, and thymus are very susceptible to the toxic action of trichothecenes; hence, various immune functions may be modulated as a result of intoxication by these mycotoxins. In mice and other animals, de-


After the observations that Canadian grains are significantly contaminated with deoxynivalenol and that this mycotoxin is carried over to finished cereal flours and foodstuffs, a guideline for deoxynivalenol in soft wheat was proposed in 1983 (Scott, 1984). These guidelines, set by the Canadian Health Protection Branch, are summarized in Table 11.16. After toxicological studies on deoxynivalenol, the tentative tolerable daily intake for adults was set at 3 µg/kg body weight, and the maximal deoxynivalenol level of 2 ppm in raw, unclean soft wheat is expected to be reduced to approximately 1.2 ppm in the flour portion of finished foods.

Table 11.16 Wheat

Canadian Guidelines for Deoxynivalenol in Soft

Unclean soft wheat Unclean soft wheat for infant foods Unclean soft wheat for bran manufacture Imported nonstaple foods

≤ 2.0 ppm ≤ 1.0 ppm ≤ 2.0 ppm ≤ 1.2 ppm (on flour or bran basis)

11.4.4

Fumonisins

The fumonisins are a family of closely related mycotoxins produced primarily by F. moniliforme and F. proliferatum. These two are the most prevalent molds associated with corn grown in all regions of the world, although they are more common in the tropical than in the temperate regions. Other Fusarium species that produce these polar metabolites include F. nygamai, F. anthophilum, F. dlamini, and F. napiforme. The fumonisins, which occur mainly in corn and in commercial corn-based human foods, are considered the aflatoxins of modern times (Scudamore, 1998). Gelderblom and associates (1988) first isolated fumonisins B1 (FB1) and B2 (FB2) from cultures of F. moniliforme MRC 826. Soon thereafter, Bezuidenhout and colleagues (1988) elucidated their structures. During the fall of 1989 and the winter of 1990, corn screenings of the 1989 U.S. crop caused numerous outbreaks of poisoning in horses and pigs. In the short time since the discovery and major outbreaks, there has been an explosion in the number of studies on these mycotoxins. The intense interest in the toxins and the fungi has arisen for a couple of reasons. First, fumonisins are found in measurable concentrations in corn grown all over the world. Second, the fungal toxins from Fusarium spp. have been epidemiologically associated with esophageal cancer in humans. These toxins cause a variety of toxic effects in various animals and probably play a role in human disease as well. Epidemiological studies suggest that fumonisins could be responsible for human esophageal cancer in South Africa and China (Norred, 1993). However, it has not been determined whether fumonisins are causally related to development of this cancer. A report from India described an acute but self-limiting food-borne disease outbreak in villagers who consumed moldy corn containing up to 64.7 mg fumonisins/kg body weight (Bhat et al., 1997). It is not known whether lower mycotoxin concentrations, chronically consumed, cause other detrimental effects in humans. Occurrence in Food Most corn and corn-containing foods and feeds are likely to be contaminated with fumonisins at detectable levels. The degree of contamination is highly dependent on environmental conditions. Environmental stresses such as heat and drought may be factors that increase the colonization of the Fusarium spp. and their production of fumonisins. Sound whole corn kernels may contain 6–10 mg/kg fumonisins; as much as 63–140 mg/kg has been detected in moldy corn (Rheeder et al., 1992). Foods with substantial


contamination (>250 µg/kg) include corn meal, corn bread mix, corn grits, and corn flour. The greatest likelihood of high levels of occurrence (>5 mg/kg) is in animal feeds, particularly those containing damaged corn and corn crack-out, such as corn screenings. Chemical Characteristics Contrary to most of the other mycotoxins, the fumonisins do not have cyclic structures. Their structures are based on a long hydroxylated hydrocarbon chain (Figure 11.26). Two hydroxyl groups are esterified to two propane-1,2,3tricarboxylic acids. FB1 differs from FB2 in that it has an extra hydroxyl group at the 10 position. Some 11 fumonisins have thus far been identified; FB1 and FB2 are the most commonly found in moldy corn. Fumonisins contain four free carboxyl groups and an amino group. Their physical structures make these toxins highly water-soluble. In fact, their insolubility in many organic solvents partly explains the difficulty in their original identification. Fumonisins are quite stable and are not broken down by moderate heat (Bordson et al., 1995). However, no fumonisins were detected in tortilla flour made by treatment with calcium hydroxide (nixtamalization), and it has been suggested that this process degrades fumonisins (Sydenham et al., 1991). However, caution is required as the breakdown products of fumonisins are just as toxic as the parent compounds (Hopkins and Murphy, 1993). Toxicological Characteristics Exposure of animals to feedstuffs naturally contaminated with fumonisins has been clearly identified as the causative agent in the incidence of equine leucoencephalomalacia (ELEM), an abnormality of the brain in horses. It has subsequently been established experimentally, by using cultures of F. proliferatum containing principally FB2 or FB3 at levels of 75 mg/kg diet fed to ponies. ELEM was observed after 150 days of feeding. The liver was also affected (Marasas et al., 1988; Bane et al., 1992). Affected animals commonly lose appetite, become lethargic, and experience neurotoxic effects after a period of ingesting contaminated feed. Autopsy showed edema in the brain and liquefaction of areas within the cerebral hemispheres. In severe cases, gross liver lesions may be seen with fibrosis of centrilobular areas. In pigs, fumonisins cause pulmonary edema and hydrothorax; thoracic cavities are filled with a yellow liquid (Bane et al., 1992). There may also be respiratory problems and fetal mortality. In chickens, fumonisins cause the so-called spiking disease characterized by neurotoxic effects in young chickens that are receiving a changing diet (Ledoux et al., 1992).

COOH

O

Fumonisin B1

COOH O

O

O

NH2

OH

OH

OH

COOH

COOH

Fumonisin B2

COOH

O

COOH O

O

O

H

NH2

OH

OH

COOH

COOH

Figure 11.26

Chemical structures of fumonisin B1 and B2.

On a weight basis, fumonisins are actually far less acutely toxic than aflatoxins. However, fumonisins commonly occur in concentrations of parts per million in corn (Shephard et al., 1996), at up to 300 mg/kg (Fazekas and Tothe, 1995), whereas aflatoxins are usually measured at concentrations of parts per billion (micrograms per kilogram) in foods. On the basis of animal data, mainly involving feeding to rats, fumonisins do not appear to be strong initiators of carcinogenesis. When rats were fed material from F. moniliforme cultures, primary hepatocellular carcinomas were produced (Gelderblom et al., 1988; Gelderblom and Snyman, 1991). A level in the diet of 250 ppm fed for 3 weeks was necessary to initiate cancer. These results were later reproduced using purified fumonisins, FB1, FB2, and FB3 (Gelderblom et al., 1993, 1994). However, experimental carcinogenicity studies have been hampered by lack of pure standards.


In longer-term feeding studies with fumonisins, the development of hepatocarcinogenesis in rats is accompanied by a significant incidence of cholangic carcinoma (Gelderblom et al., 1991). Since hepatocarcinogenesis in rats by fumonisins is accompanied by toxic hepatitis and cirrhosis, it has been suggested that although poor initiators, fumonisins may be more powerful promoters, acting at dietary levels much lower than those required for initiation and complete carcinogenesis. This would correlate with the fact that fumonisin test results have been found to be negative in Ames tests (Park et al., 1992), in vitro rat hepatocyte DNA repair assay results (Gelderblom et al., 1989), and micronucleus test results (Gelderblom et al., 1995). The IARC (1993) concludes that there is limited evidence in experimental animals for the carcinogenicity of FB1 and that toxins derived from F. moniliforme are possibly carcinogenic to humans (group 2B). More extensive

studies are required to generate a better picture of the chronic toxicity of the fumonisins. Lebepe-Mazur and coworkers (1995a, 1995b) showed that FB1 affected the fetus in pregnant rats, causing low litter weight and fetal bone development as compared with those of controls. Developmental toxicity has also been noted in Syrian hamsters (Floss et al., 1994). Flynn and associates (1996), on the basis of studies with cultured rat embryos, concluded that rat embryos are highly sensitive to FB1 toxicity. No definitive studies have been done to establish conclusively whether fumonisins are developmental or reproductive toxins in farm animals or humans at the levels encountered in naturally contaminated foodstuffs. Human exposure to fumonisins through the ingestion of contaminated food has been estimated in several countries. In Canada, between 1991 and 1995, consumption averaged about 0.109 µg/kg/day (Kuiper-Goodman et al., 1995), and in the Transkei, South Africa, 14 µg/kg/day (Thiel et al., 1992). Despite very high levels of human exposure to these mycotoxins, there are no details in the literature of any resulting toxicities. It appears, therefore, that although there are animal data of toxicities at low levels of exposure, humans are not very sensitive to the acutely toxic effects of fumonisins. One of the principal concerns in terms of hazards to human health, however, as mentioned earlier, is the possible involvement of exposure to fumonisins in the development of esophageal cancer. Such a possibility has been indicated by epidemiological studies in South Africa, where there are high incidences of esophageal cancer in areas of high contamination of the staple foodstuff, corn, with fumonisins (Rheeder et al., 1992). In these areas, contamination of food with other mycotoxins is also quite low. Studies in areas of China with high incidences of esophageal cancer have shown correlations with exposure to fumonisins (Chu and Li, 1994). In certain areas of northern Italy, where there is one of the highest incidences of esophageal cancer in Europe, locally grown corn forms a substantial portion of the diet. It has been found to be contaminated with fumonisins and possibly to constitute a significant risk factor to human health (Visconti et al., 1996). There is a strong structural similarity between FB1 and sphinganine. This has led to the hypothesis that fumonisins exert their toxic effects through a disruption of sphingolipid metabolism or inhibition of a function of sphingolipids (Riley et al., 1994). The fumonisins are potent inhibitors of sphinganine N-acetyl transferase, an enzyme involved in sphingolipid biosynthesis and turnover (Figure 11.27). Sphingolipids have important roles in membrane and lipoprotein structure, cell-to-cell communication, interaction between cells and the extracellular matrix, and regulation


of growth factor receptors and as second messengers for a wide range of factors, including the tumor necrosis factor and interleukin-1. The resulting accumulation of free sphinganine is growth-inhibitory and cytotoxic to cells. The loss of complex sphingolipid synthesis would be expected to alter cell behavior and could also lead to cell death, on the basis of findings with mutants in serine palmityltransferase, the initial enzyme of the sphingolipid biosynthesis. Regulation The U.S. FDA has utilized informal guidance levels for fumonisins in animal feed since 1993. The levels proposed earlier were based on those recommended by the Mycotoxin Committee of the American Association of Veterinary Laboratory Diagnosticians (AAVLD). The U.S. FDA had not determined a definitive “safe level” for fumonisin residues at that time because the then-available data did not provide enough information to allow determination of a safe level for the toxin residues in milk, meat, and eggs. The agency concluded that limiting exposure to fumonisin in animal feed will protect consumers from unsafe residues, on the basis of the following toxicological and biochemical properties of fumonisins and the metabolism studies conducted to date: 1.

2.

3.

Fumonisins, which are water-soluble, do not bioaccumulate in animal tissues and thus are less likely to leave residues than lipid-soluble mycotoxins. Because fumonisins are structurally related to endogenous substrates (sphinganine and sphingosine), the mycotoxins are susceptible to be detoxified into less toxic metabolites. Residues exist usually in organ tissues, kidney and liver, which humans consume less than muscles.

These guidance levels for fumonisins in livestock feed were as follows: horse (nonroughage portion), 5 ppm; porcine (total ration), 10 ppm; and poultry (total ration) and beef cattle (nonroughage portion), 50 ppm total fumonisins. In June 2000, the U.S. FDA revised guidelines for industry for fumonisin levels in human foods and animal feeds. These guidelines are considered adequate to protect human and animal health and, according to the agency, are achievable in human foods and animal feeds with the use of good agricultural and good manufacturing practices. The revised guidelines are summarized in Table 11.17. Tolerance levels for fumonisins have not been set for foods intended for human consumption in most other countries. However, Switzerland has set a level of 1 ppm fumonisins in cereal grains.

Palmityl CoA + Serine

Sphinganine N-Acyltransferase Fatty acyl CoA

Ceramide N-Acyltransferase

Sphingolipid turnover products

Fatty acyl CoA

Sphingosine

Site of inhibition by fumonisins Figure 11.27

The disruption of sphingolipid pathway by fumonisins.

Table 11.17

U.S. Food and Drug Administration Guidance Levels for Fumonisins in Human Foods and Animal Feeds

Foods Human foods Degermed dry milled corn products (e.g., flaking grits, corn grits, corn meal, corn flour with fat content 20 1.5 1.5 4.5

Source: Compiled from Concon (1988) and Antkowiak (1996).


C. splendens were reported from France (Gerault, 1981). Schumacher and Hoiland (1983) have extensively reviewed several cases of Cortinarius spp. poisoning from various countries.

Unlike the well-documented clinical symptoms and histopathological changes caused by the ingestion of Cortinarius spp. mushrooms, the mode of action of their toxins is still not fully elucidated. There is a relationship between the structure and physiological activities of orellanine and two biologically active cationic bipyridines with wellknown herbicidal properties, diquat (N,N′-dimethyl-2,2′bipyridine) and its 4,4′-isomer, paraquat. Schumacher and

HO

HO

HO

O

HO

OH

OH N

N

N

OH

OH

OH (a) Orellanine

HO

O

OH

O

N

N

N HO

(b) Orellinine

(c) Orelline

Figure 15.4 Toxic principles of Cortinarius orellanus mushroom.

Hoiland (1983) suggested a similar pathogenic mechanism of action for all these compounds, assuming their ability to inhibit the intracellular metabolic systems dependent on reduced oxidized nicotinamide-adenine dinucleotide (NADPH) plus H+, by participation in the electron transport process (Figure 15.5). Additionally, the concomitantly produced superoxide also has cytotoxic properties, either direct or by generation of other reactive forms of oxygen such as singlet oxygen. This proposed intracellular toxic mechanism with NADPH plus H+ depletion would need a long time to impoverish the cells to the degree of inevitable necrosis. This is in accordance with the delayed toxic effects observed in this type of mushroom poisoning. This hypothesis, although convincing, has not been con-

firmed by the studies of the electrochemical behavior of orellanine, which was shown to be different from diquat and paraquat (Richard et al., 1988). Rapior (1988) suggested that the Cortinarius spp. toxicity is caused by metabolites with the isoxazolium core derived from the photochemical rearrangement of orellanine. These intermediates can bind covalently with numerous proteins in the body, leading to organ damage. Rapior (1988) also found that orellanine purified in the dark and administered to laboratory animals showed low toxicity, whereas that extracted in the light induced a toxic response. These observations indicating the consequence of photoexcitation supported the proposed phototoxicity mechanism of orellanine (Figure 15.6).

OH N Enzyme reduced form

O

OH

HO

O22–

O N

HO

OH N O Enzyme oxidized form

HO

O N

HO

Figure 15.5 Intracellular toxic mechanism proposed for orellanine.


OH O2

Orellanine

Orellinine

HO

Orelline

HO

HO N

N HO

O

O

N

HO

HO OH

O

OH

OH

N

N

N

OH

OH

hν

OH hν

hν

HO

hν

HO N

HO

N O

O OH

O N OH

Intermediates capable of binding with proteins

N OH

Figure 15.6 Photodecomposition of orellanine.

The LD50 values of orellanine are 4.9, 8.3, and 8.0 mg/ kg in the cat (per os), mouse, and guinea pig (parenteral), respectively. These values and the symptoms observed in victims indicate the extreme toxicity of this compound (Concon, 1988). 15.5.3

Gyromitrin

Gyromitrin poisoning is associated predominantly with the consumption of Gyromitra esculenta, the false morel. This mushroom was considered a delicious edible mushroom for centuries until it turned out to contain, in the raw state, toxic substances that can cause death. Franke and associates (1967) presented a compilation of 513 cases of poisoning including 14.5% fatalities. The severe cases occurred after ingestion of raw or insufficiently cooked false morels, whereas after extensive cooking and discarding of the water or after drying of the mushrooms, poisoning occurred only rarely. Intoxications resulting from the ingestion of this mushroom are seen most frequently in Eastern Europe and only occasionally elsewhere. In Poland, it is the third most common poisoning after those caused by A. phalloides and Paxillus involutus (Antkowiak, 1996).


Chemical Characteristics The toxic properties of G. esculenta and other related species result from the presence of harmful hydrazine derivatives in the mushrooms. The main component, gyromitrin (2-ethylidene-1-formyl-1-methylhydrazine, Figure 15.7a) is a volatile compound. It is an unstable compound, prone to hydrolysis and oxidation. On acid hydrolysis in the GI tract, gyromitrin is converted first to 1-formyl-1-methylhydrazine (Figure 15.7b), which after the loss of the formyl residue yields the highly toxic N-methylhydrazine (Figure 15.7c). Additional homologs of gyromitrin, in which the acetaldehyde is replaced by other aldehydes, now also are known to exist. These include the derivatives of pentanal, 3-methylbutanal, and hexanal. All of these compounds readily hydrolyze to form the active toxin, monomethylhydrazine. The toxin is volatile and water-soluble and thus can be eliminated from the mushroom by air-drying or extraction with boiling water. The content of gyromitrin in dried mushrooms is about 3 mg/kg, compared to 1.2 to 1.6 g/kg in fresh mushrooms. The instability of gyromitrin suggests that in the mushroom it is probably bound to high-molecular-weight components, possibly as glycoside (Stijve, 1978).

CH3 H3C

CH

N

CH3 H2N

N CHO

(a) Gyromitrine

CH3 H2N

N CHO

N H

(b) N,N-Formylmethylhydrazine

(c) N-Methylhydrazine

Figure 15.7 Chemical structures of Gyromitra esculenta toxins.

Symptoms of Poisoning The symptoms of gyromitrin poisoning are similar to those caused by cyclopeptide poisoning. They appear suddenly, usually 6 to 12 hr after ingestion or inhalation of the vapor from cooking mushrooms. They are characterized by nausea, vomiting, abdominal pain, dizziness, headache, abnormal thirst, general weakness, and painful liver enlargement, but only exceptionally accompanied by diarrhea. In most cases this phase ends with recovery within 2–6 days. However, after ingestion of large amounts of weakly cooked false morels, a hepatorenal phase can follow with symptoms of liver injury, hemoglobinuria, or anuria. Predominant symptoms of this phase are nervous disturbances such as difficulties in moving, cramps, deliria, and even unconsciousness. Death can occur through collapse of circulation (Faulstich and Wieland, 1992). The treatment for gyromitrin poisoning is the same as for isoniazid overdose and consists of administering pyridoxine and correcting the systemic acidosis. In the case of jaundice and coma, it is the same as for phalloides poisoning. The monomethyl derivative of gyromitrin appears to be neurotoxic and carcinogenic, causing liver and intestinal tumors in animals. It is also shown to be embryotoxic and teratogenic (Slanina et al., 1993).

15.6 NEUROTOXIC MUSHROOMS The symptoms caused by neurotoxic mushrooms may occur almost immediately from 15 to 180 min after ingestion. The composite symptoms as observed in various cases of poisoning of this type include increased salivation, lacrimation, perspiration, and severe gastrointestinal disturbance with vomiting and copious water diarrhea. The pulse becomes slow and irregular (Concon, 1988). There may be labored or asthmatic breathing, vomiting, hallucinations, and confusion. Well-known mushroom toxins belonging to this group are described in the following sections.


15.6.1

Muscarine

Muscarine certainly is the most famous mushroom poison. In Europe, muscarine intoxication is most often caused by Inocybe patouillardii, commonly known as red-staining inocybe. This mushroom is often confused with the edible Tricholoma georgii or Agaricus campestris. Muscarine was long considered to be the toxin produced by all poisonous mushrooms (Schmiedeberg and Koppe, 1869). The toxin was first isolated from Amanita muscaria, from which the name of the toxin originated. This mushroom, however, contains muscarine in only traces, whereas muscarine is found in large amounts in mushrooms of the genera Clitocybe and Inocybe. Almost all Inocybe species contain the toxin and hence should be avoided. The most popular, I. patouillardii, is definitely known to cause death. It contains 370 mg/kg muscarine, relative to the fresh weight of the fruit body, as compared to 2–3 mg/kg in A. muscaria. Additionally, low concentrations (≤ 2 mg/100 g dry weight) of muscarine and its stereoisomers have been found in the fruiting bodies of the following species of agaricales: A. pantherina, A. phalloides, Boletus calopus, B. luridus, Clitocybe dealbata, C. hydrogramma, C. gibba, C. vermicularis, Clitopilus intermedius, Collybia peronata, Hygrocybe nigrescens, Hypholoma fasciculare, Lactarius rufus, L. trivialis, Mycena pelianthia, M. pura, Paxillus involutus, Rhodophyllus rhodopolius, R. sinuatus, Russula emetica, Tricholoma sulphureum, and Tylopilus felleus (Antkowiak, 1996). Chemical Characteristics Muscarine is 2-methyl-3-hydroxy-5-trimethylammoniummethyl-tetrahydrofuran. Eugster (1956a, 1956b) and simultaneously Kogl and colleagues (1957) revealed its complete chemical structure by using x-ray diffraction techniques. The compound has three chirality centers, and hence, four pairs of enantiomers are possible. All diastereomers: (+)-(2S,3R,5R)-muscarine, (–)-(2S,3R,5R)-allomuscarine, (+)-(2S,3S,5S)-epimuscarine, and (+)(2S,3S,5R)-epiallomuscarine (Figure 15.8a–d), can occur

HO

HO 3

3

4

2

5

H3C

2

N(CH3)3 H3C

1 O

(a) L-(+)-Muscarine (2S, 3R, 5S)

5

N(CH3)3

1 O

(b) L-(+)-Epimuscarine (2S, 3S, 5S)

HO

HO 3

4

2 H3C

4

5

N(CH3)3

N(CH3)3

H3C

1 O

O

(c) L-(-)-Allomuscarine (2S, 3R, 5R)

(d) L-(+)-Epiallomuscarine (2S, 3S, 5R) O N(CH3)3

H3C

O (e) Acetylcholine

Figure 15.8 Diastereomeric muscarines and their structural similarity with acetylcholine.

in nature. The physiological activity of the stereoisomers differs; for natural muscarine it is 100-fold stronger than that of the others, whereas D-(–)muscarine is only 10–3 as active as the naturally occurring enantiomers. Mechanism of Action Through its structural similarity to acetylcholine (Figure 15.8e), muscarine binds to the acetylcholine receptor on synapses of the nerve endings of smooth muscles and endocrine glands, causing the well-known parasympaticomimetic effects. However, it is more selective. Muscarine binds strongly with the muscarine type of acetylcholine receptors in smooth muscles of the GI tract, eye, exocrine glands, and heart; acetylcholine exerts an additional “nicotinic action” by affecting ganglia and voluntary muscles. Because muscarine is not an ester like acetylcholine and hence resists esterase activity, it is not degraded but can cause continuous stimulation of the affected neurons (Faulstich and Wieland, 1992). The binding affinity of muscarine with the receptors is so high that the muscarine stereoisomers are routinely used to study cholinergic pharmacological characteristics. The cholinomimetic effects of muscarine are successfully antagonized by atropine. The involvement of muscarine in


the phospholipid metabolism in ganglia has also been shown (Horwitz et al., 1986). The selective binding affinity of stereoisomers of muscarine strongly depends on even small changes in the structure, conformation, and configuration of the rigid muscarine molecule. The hydroxyl group trans-arranged to the other two substituents plays a key role (Antkowiak, 1996). The change of the CH-OH group into carbonyl yields muscarone, which is more potent than muscarine but less selective. As does acetylcholine, it exhibits strong stimulating and blocking effects on ganglionic synapses and neuromuscular junctions. Symptoms of Poisoning The symptoms of parasympathetic stimulation evoked by muscarine are usually evident within 15 to 30 min after ingestion. The toxic response is not affected by cooking. There appears to be a dose-response relationship as to the appearance of any particular effect, the most sensitive of which is profuse sweating. As the quantity of muscarine increases, one also may observe nausea, vomiting, and abdominal pain. More severe intoxications produce blurred vision, salivation, lacrimation, rhinorrhea, and diarrhea. Tremors, dizziness, and bradycardia are only rarely seen.

Death may occur in a few hours unless the poisoning is diagnosed and promptly treated with atropine. Atropine competes with muscarine for binding to the receptor sites but does not evoke a signal. In mice, muscarine has an LD50 of approximately 0.25 mg/kg body weight when administered intraperitoneally, and higher if administered orally (Faulstich and Wieland, 1992). Even if humans were several times more sensitive to muscarine than mice, severe intoxication of humans would require a large amount of A. muscaria. 15.6.2

Muscimol and Ibotenic Acid

The toxins muscimol and ibotenic acid cause the so-called pantherine syndrome, which is attributed to the consumption of Amanita muscaria (fly agaric), A. pantherina (panther cap), and A. strobiliformis (grows only in Japan) mushrooms. The symptoms of illness occurring after the ingestion of these mushrooms cannot be attributed to muscarine, which is present only in trace amounts. Numerous reports of poisoning after the consumption of these mushrooms for food are documented in the older literature. Two

Chemical Characteristics The toxicity of these mushrooms is attributed to two isoxazoline compounds, ibotenic acid (also known as pramuscimol, α-toxin) and muscimol (initially also called pantherine, pyroibotenic acid, β-toxin, or agarin). In addition, an oxazolone, muscazone, has been isolated from these mushrooms. Both ibotenic acid and muscimol appear to be limited to these three mushroom species only. Ibotenic acid is α-amino-(4-hydroxy-isoxazole-2)-yl-acetic acid, a structure related to and possibly derived from βoxo-L-glutamine (Figure 15.9a). Muscimol (Figure 15.9b) is the decarboxylation product of ibotenic acid. Muscazone (Figure 15.9c), found only in A. muscaria collected in summertime, appears to be formed from ibotenic acid by a photoinduced rearrangement (Goth et al., 1967). Tricholomic acid (Figure 15.9d) is a related isoxazole found only in Tricholoma muscarium. This mushroom is found only in Japan.

(a)

HO

Ibotenic acid

medium-sized mushrooms of A. muscaria and only one of A. pantherina are sufficient for intoxication of an adult.

(b) O Muscimol

N

NH3

N

O

NH3 O

COO

(d)

(c) O

HN

Tricholomic acid

H

Muscazone

NH3 O

H HN

NH2

O O

COO

COOH (e)

(f)

HO

O NH3

O

NH3 O

COO Glutamic acid

γ-Aminobutyric acid (GABA)

Figure 15.9 Ibotenic acid and muscimol as analogs of glutamic acid and GABA along with related toxins.


The amounts of the isoxazoles present in these mushrooms are approximately 0.2% for dried A. muscaria and 0.4% for A. pantherina.

a source of muscimol that is generated in the body by decarboxylation.

Symptoms of Poisoning

15.7 HALLUCINOGENIC MUSHROOMS

These mushroom toxins exhibit a powerful action on the central nervous system, causing atropine-type symptoms. When they are eaten in large amounts, the poisoning symptoms may appear as early as 15 to 30 min; however, usually they occur 1 to 3 hr after ingestion. The initial weakness, balance disorder, sweating, and gastrointestinal discomfort resembling advanced alcoholic intoxication are followed by anxiety, dizziness, elation, increased motor activity, tremors, illusions, and even maniac delirium. This may alternate with periods of drowsiness or sleep, after which the awakened patient has no memory of his or her experience (Antkowiak, 1996). Increased pulse rate and body temperature up to 40°C (104°F) may be observed. In severe cases, unresponsive, maximally dilated pupils may also be observed. Finally, coma accompanied by cardiovascular collapse and respiration depression may occur. However, in the majority of cases, symptoms abate rapidly, usually after approximately 24 hr. In the past, these mushrooms were eaten deliberately in several European countries in order to produce hallucinogenic effects. Incidences of poisoning have been increasing in the United States, Europe, and South Africa, especially in children. Mechanism of Action The neurotransmitting properties of ibotenic acid and muscimol are linked to the structures of these alkaloids, which resemble those of some amino acids of the nervous system. They reveal the powerful properties of glutamate (Figure 15.9e) and γ-aminobutyric acid (GABA) (Figure 15.9f) agonists. In fact, ibotenic acid and muscimol may be considered as conformationally restricted analogs of these amino acids, binding alternatively to their respective receptors (Gore and Jordan, 1982). Baldelli and coworkers (1994) have shown that GABAA receptors on rat cellular granule cells are even more potently activated by muscimol than by GABA itself in inducing Cl– currents. Both ibotenic acid and muscimol have been suggested as possible models for the development of potent drugs in the treatment of specific nervous diseases. In rats the LD50 of muscimol was determined to be 45 mg/kg by oral and about 4.5 mg/kg by intravenous administration; the corresponding values for ibotenic acid are 129 and 42 mg/kg, respectively (Theobald et al., 1968). Perhaps ibotenic acid exerts its toxic effects only as


Besides Amanita muscaria and A. pantherina, which cause sedative-hypnotic actions due to the presence of the toxic isoxazole alkaloids, a few other mushrooms with such hallucinogenic properties are known. The physiologically active substances responsible for such symptoms are 4- or 5substituted tryptamines that may be represented by psilocybin and bufotenine, respectively. Striking features of the symptoms produced by hallucinogenic mushrooms are the hallucinatory or psychedelic effects and, depending on the species, prolonged euphoria and excitation. The effects may be accompanied by other serious symptoms such as muscular incoordination and weakness of the arms and legs, sometimes with complete but temporary paralysis. The unpleasant effects may last for several hours. Mushrooms containing hallucinogenic indole derivatives are found in the genera Psilocybe, Panaeolus, Pholiotina (Conocybe), Gymnopilus, and possibly Panaeolina. Psilocybe semilanceata is widely distributed in Europe and North America. 15.7.1

Psilocybin and Psilocin

Psilocybin and its accompanying congener psilocin were first isolated from Psilocybe mexicana and identified as active compounds of this hallucinogenic mushroom by Hofmann and associates (1958a, 1958b; Hofman, 1959). Like the mycotoxin ergot alkaloids (Chapter 11), psilocybin and related alkaloids have a 3,4-distributed indole ring system, though the structure is less complicated. Psilocybin is a derivative of tryptophan; its chemical structure is 4-phosphoryloxy-dimethyltryptamine or 4-phosphoryloxy-3-[2-(dimethylamino)-ethyl]-indole (Figure 15.10a). The dephosphorylated compound, psilocin (Figure 15.10b), is not hallucinogenic and, unlike the phosphoric ester, easily decomposed by oxidation. In addition to these two compounds, baeocystine (Figure 15.10c) and norbaeocystin (Figure 15.10d) are the monomethylated and nonmethylated derivatives of psilocybin, respectively. These are found in Psilocybe baeocystis (Leung and Paul, 1968). The highest concentration (>0.5% of dry weight) of psilocybin is generally found in Psilocybe semilanceata (liberty cap mushroom), Panaeolus subbalteatus, Conocybe cyanopus, and Pluteus salicinus (Ohenoja et al., 1987). The first two are the only psilocybin containing mushrooms that can be gathered in middle and northern

(d)

O

(a)

O

P

O

O O

P

O

OH

OH CH2CH2

NH

CH2CH2NH3

CH3

CH3 N H

Norbaeocystin

N H

Psilocybin

(e) HO

(b)

CH2CH2

N

OH

CH3

CH3 CH2CH2

N

N H

CH3

CH3 N H

Bufotenine

(f) CH

Psilocin

CH

O

O

Yangonin H3CO

O

(c) O

P

OCH3

O

(g)

OH CH2CH2

NH2 CH

CH

O

O

CH3

bis-Noryangonin

N H

Baeocystin

HO OH

Figure 15.10

Structures of some hallucinogenic toxicants found in various species of mushrooms.

Europe in sufficient quantities for intoxication to occur (Stijve and Kuyper, 1985). The dried fruiting bodies of Psilocybe mexicana contain 0.2%–0.4% psilocybin and only trace amounts of psilocin. Symptoms after ingestion of about 20 g of fresh mushrooms, or 4–8 mg psilocybin, start within the first half hour. Reactions to the drug vary between individuals from a feeling of relaxation to one of tension, anxiety, or dizziness. Sometimes nausea and abdominal discomfort including vomiting and diarrhea may occur. During the second half hour the occurrence of visual effects is generally seen, including the perception of brilliant colors and objects with closed eyes. Panic reactions, such as fear of death or insanity, have also been reported. During the following hours the visual effects increase and then disappear. Systemic effects are mostly due to the stimulation of the central or sympathetic nervous system and may comprise papillary dilatation, rapid heartbeat, and high blood


pressure, combined with low blood sugar level and decreased body temperature (Vergeer, 1983; Faulstich and Wieland, 1992). Therapeutic intervention is rarely sought or required for adults. The clinical effects may be terminated, however, by the administration of diazepam or a phenothiazine. The LD50 values of psilocybin in mice are 275 mg/ kg intravenously and 420 mg/kg intraperitoneally (Antkowiak, 1996). 15.7.2

Bufotenine and Related Compounds

Bufotenine, 5-hydroxy-N,N-dimethyltryptamine (Figure 15.10e), was first identified and isolated from false death cap mushroom, Amanita mappa (identical with A. citrina), by Wieland and colleagues (1953). It is also found in other Amanita species, such as A. muscaria, A. pantherina, A. porphyrea, and A. tormentella. Stijve (1979) demonstrated

the presence of 0.03%–0.06% of another related alkaloid, bufotenine N-oxide, in A. citrina mushrooms collected in various parts of Europe. Bufotenine has always been considered a psychomimetic agent. Fabing and Hawkins (1956) gave an intravenous injection of a dose up to 16 mg of this compound to a healthy young man, which was feasible without jeopardizing his life. It resulted primarily in a burning sensation in the face, which turned purple and returned to its normal color at the end of an hour; visual disturbances (red spots passing before the eyes) accompanied by nystagmus and mydriasis, as well as a change in the perception of time and space. These effects resembled those of lysergic acid diethylamide (LSD25) and mescaline but developed and disappeared more quickly. Subsequently, Bhattacharya and Sanyal (1971) demonstrated the anticholinesterase activity of bufotenine in rats at the dose of 2.5 mg/kg given intraperitoneally. Bufotenine is capable of increasing the basal neurotransmitter efflux by blocking the neural membrane transport carrier. Its action on 5-hydroxytryptamine (5-HT) neurons is comparable to that of p-chloroamphetamine (Monroe et al., 1994). The concentrations of bufotenine and related compounds are generally too small to cause any toxicity of these mushroom species. 15.7.3

Psychoactive Effects of Gymnopilus spectabilis

In Japan, the mushroom Gymnopilus spectabilis is believed to cause hallucinations and abnormal behavior. However, this mushroom is frequently consumed without any psychoactive effects after the bitter taste is removed by boiling water. Intoxications generally occur as a result of ingestion of inadequately cooked mushrooms (Kusano et al., 1986). The hallucinogenic effects of this mushroom are attributed to yangonine (Figure 15.10f) and bis-noryangonin (Figure 15.10g). Unlike the other hallucinogenic principles in mushrooms, which are all tryptamine derivatives, the yangonines are styrylpyrones.

15.8 MUSHROOM TOXINS AFFECTING THE HEMATOLOGICAL SYSTEM 15.8.1

Mushrooms with Disulfiramlike Activity (Coprinus Syndrome)

Several mushroom species are capable of inducing a marked sensitivity to alcohol. This effect is encountered most commonly with Coprinus atramentarius (common inky cap mushroom), which is a particularly desirable edi-


ble mushroom. However, if alcohol is consumed up to approximately 72 hr after the ingestion of this species, the individual may experience flushing, hypotension, tachycardia and palpitations, paresthesias, severe nausea and vomiting, and an intense headache (Faulstich and Wieland, 1992). The effects normally disappear after 3–4 hr, but occasionally may last up to 50 hr. These effects are similar to those caused by disulfiram (Antabuse), employed in the treatment of alcoholism. Therapeutic intervention usually is not required. The agent causing these symptoms has been identified as coprine, N5-(1-hydroxycyclopropyl)-L-glutamine (Figure 15.11). This phototoxin was isolated almost simultaneously in the United States (Hatfield and Schaumberg, 1975) and in Sweden (Lindberg et al., 1975). This substance is converted in the body into 1-aminocyclopropanol (Figure 15.11) and thence to the active toxin, 1-hydroxycyclopropylammonium ion (Figure 15.11). The active toxin reversibly inhibits the low-Km acetaldehyde dehydrogenase of the liver by blocking an essential sulfhydryl group of the enzyme (Wisemann and Abeles, 1979). This results in the accumulation of acetaldehyde in the blood during alcohol metabolism. The elevated acetaldehyde levels are primarily responsible for the intoxicating symptoms described. The coprine content of fresh C. atramentarius mushroom is about 160 mg/kg (Lindberg et al., 1977). Coprine is also found in C. quadrifidus, C. variegates, and C. insignis. Other mushrooms producing similar alcohol-sensitizing activity include Boletus luridus, Clitocybe clavipes, and Verpa bohemica; however, none of them has been found to contain coprine.

H2C

OH C

H2C

NH

NH3

O C

CH2

CH2

CH

COO

Coprine

H2C

OH

H2C

NH2

H2C

C

C H2C

1-Aminocyclopropanol

O

Cyclopropanone

Figure 15.11 Chemical structure of coprine and its conversion to the toxic metabolite cyclopropanone.

15.8.2

Mushrooms Causing Hemolysis

Hemolysis-causing agents have been found in several mushroom species, including the quite popular Agaricales spp. and rarely in Boletaceae and Russula spp. These toxins have been also detected in the species from the families Hygrophoraceae and Strophariaceae, and from genera Mycena, Oudemansiella (Tricholomataceae), Hebeloma, Gymnopilus (Cortinariaceae), and Amanita. The latter genus includes species such as A. muscaria (causes agglutination of red blood cells), A. gemmata, A. verna, A. citrina, A. porphyria, A. spissa, A. echinocephala, and, invariably, A. rubesscens and A. phalloides, which contain high activity (Seeger and Widemann, 1972; Seeger et al., 1973; Antkowiak, 1996). Although in most cases, the chemical identity of the toxin(s) has not yet been determined, the principal hemolytic activity in A. phalloides is attributed to phallolysin. It appears to be a mixture of two to three cytolytic proteins. The hemolytic activity of phallolysin results in the release of hemoglobin from erythrocytes into the blood plasma, and its toxicity exceeds even that of α-amanitin severalfold (Faulstich et al., 1983). Although the sensitivity of mammals to phallolysin increases with body size (LD50 intravenously in mice is 120 µg/kg, in rats is 50 µg/kg, and in rabbits is even less than 25 µg/kg) and death occurs by acute hemolysis only a few minutes after intravenous or intraperitoneal application, it still cannot contribute to human intoxications. This is because phallolysin, as a protein, is unstable when heated over 65°C or when treated with acid, which should cause the inactivation of the toxin on contact with the gastric juice (Wieland, 1986). 15.8.3

The Paxillus Syndrome

The paxillus syndrome is associated with the consumption of Paxillus involutus, commonly known as brown roll-rim mushroom, and Suillus luteus. Both are abundant and sometimes consumed in large quantities. If not thoroughly cooked before eating, these mushrooms very often have been the cause of intoxications, sometimes leading to severe consequences. The symptoms of poisoning occur within 1–2 hours after ingestion and include intense abdominal pain, nausea, vomiting, diarrhea, and acute circulatory collapse, followed by oliguria and hemoglobinuria. Schmidt and coworkers (1971) reported on a hemolytic anemia that developed after eating of P. involutus followed by a massive hemolysis with subsequent shock and acute renal failure that occurred after renewed consumption of this mushroom. According to Winkelmann and associates (1986), fatal poisoning with P. involutus is caused by an immune reaction in which immune complexes at-


O OH

HO HO

HO

OH

Figure 15.12 Chemical structure of involutin, the toxic secondary metabolite of Paxillus involutus mushroom.

tached to the erythrocyte surface initiate hemolysis. Therefore, in addition to adequate shock treatment, elimination of these immune complexes by plasma separation seems to be the therapy of choice. Edwards and colleagues (1967) isolated the only secondary metabolite of P. involutus not found in other mushrooms, called involutin (Figure 15.12). However, there is no evidence that this nitrogen-free compound may be the toxin. 15.8.4

Mushrooms with Hemagglutinating Activity

Lectins or hemagglutinins are present in the aqueous extracts of many species of various families including Hygrophoraceae, Tricholomataceae, Agaricaceae, Strophariaceae, Cortinariaceae, and Russulaceae, provided they are collected no earlier than September (Seeger and Wiedemann, 1972). These carbohydrate-binding proteins, which cause agglutination of red blood cells, have been purified from a variety of mushrooms, such as Agaricus bisporus, A. campestris, Clitocybe nebularis, Marasmius oreades, Fomes fomentarius, Flammulina velutipes, Psathyrella velutina, Grifola frondosa, and Aleuria aurantia (Antkowiak, 1996).

15.9 MUSHROOMS CAUSING GASTROINTESTINAL DISTURBANCES Although poisoning that produces gastrointestinal symptoms has been estimated to amount to up to 40% of all cases of mushroom poisoning (Alder, 1960), reports are not numerous because fatalities are quite rare, and the patients normally recover within a few days. In the few casualties reported so far, only children were involved (Chapuis, 1984). A large number of widely distributed mushrooms are capable of causing gastrointestinal irritations (Table 15.1). Usually symptoms occur 15 min to 3 hr after the mushroom meal. Depending on the mushroom, the intoxi-

33H16C

HOOC

29H14C

COOH

CH

C

CH2

HOOC

COOH

COOH C

CH

CH2

COOH

OH

OH Norcaperatic acid

Agaricic acid

O

N

H N

N

COO N H

N H

O

NH3

Agaricone

HO

OCH3

OH

OH

Xanthodermin OH CH3

N

OHC

CH3

CH3

N Agaricin

Figure 15.13

COOH

N

N

SO3Na

CH3

HO

O

O

N Psalliotin

Illudinine

Illudalic acid

Chemical structures of some gastrointestinal irritants found in various mushrooms.

cation may produce persistent nausea, vomiting, diarrhea, and abdominal pain, which may lead to dehydration and hypovolemic shock, especially in children. The importance of fluid and electrolyte replacement in children must be emphasized. Mushrooms eaten by adults almost invariably have been cooked, by a process that markedly reduces or even inactivates some gastroenteric irritants. Many childhood fatalities that follow the ingestion of normally “nontoxic” mushrooms are secondary to uncompensated fluid and electrolyte losses resulting from profound emesis and diarrhea in response to gastroenteric irritants in the raw mushrooms. Otherwise, the management is entirely symptomatic as for gastritis of any other cause. Unlike in the cases of well-known fatal consequences of ingestion, most of the mushrooms have not been thoroughly investigated yet with regard to their poisonous principles and pathogenic effects. The chemical structures of some of the gastrointestinal irritants identified thus far are shown in Figure 15.13.

15.10 MUSHROOMS WITH OBSCURE TOXICITY PATTERN Several species of mushrooms can only be listed as “suspected” because there are insufficient clinical case re-


ports to have attracted the attention of investigators. In addition to the possibility of obtaining a specimen of the offending mushroom for identification, there is the problem of the great variability in toxicity. Thus a species growing in one part of a country may produce sickness, whereas in another section of the same country it may be eaten with relish. Part of this is due to variability in growing conditions. Certain mushrooms are also capable of forming hybrids. Hypholoma fasciculare (Naematoloma fasciculare), for example, seems to be edible, although not particularly desirable, in some locations and dangerously toxic elsewhere. In a few cases of fatal intoxications by this mushroom studied by Herbich and colleagues (1966), about 9 hr after the mushroom meal gastrointestinal symptoms occurred. These were, however, preceded by extensive liver damage that later resulted in death. Ikeda and coworkers (1977a, 1977b, 1977c) isolated two groups of active compounds from these mushrooms, fasciculols (Figure 15.14) and naematolins (Figure 15.15). Fasciculols are hydroxylated tetracyclic triterpenes of a common lanostane skeleton with one tetrasubstituted double bond. They are rare natural compounds containing two α-glycol systems at the positions C-2,C-3, and C-24,C-25. Naematolin is a sesquiterpenoid with a caryophyllane bicycloundecane skeleton.

R4 H

OH

2HC

R3 OH

R1O

R2O

O

OH

H

O

O

OH

OCH3

OH

N H

(Y)

O

(X)

O

O

OH

O OH N H O

(Z)

Fasciculol A Fasciculol B Fasciculol C Fasciculol D Fasciculol E Fasciculol F Fasciculic acid A Fasciculic acid B Fasciculic acid C

Figure 15.14 mushroom.

R1

R2

R3

R4

H H X H H X Y Y H

H H H H X H H H Z

H OH OH OH OH OH H OH OH

H H H OH OH OH H H OH

Active hepatotoxic compounds of fasciculol type isolated from Naematoloma fasciculare (Hypholoma fasciculare)

Yet another example is the ugly milk cap mushroom, Lactarius necator (synonyms L. plumbeus, L. turpis), which is widely distributed in Europe, Asia, and North America. Studies on the chemical composition of this mushroom by Suortti (1984) and Suortti and von Wright (1983) led to the identification of necatorin, the activity of which appeared to be comparable with that of aflatoxin B1 in the microbiological mutagenicity tests. Several other alkaloids were also found in this mushroom (Figure 15.16). The commercially cultivated Agaricus bisporus mushroom was found to contain several phenylhydrazine


and aniline derivatives of potential toxicity. Both have been implicated in carcinogenesis. The well-characterized compound of this class is agaritine. Agaritine is hydrolyzed in the mushroom by γ-glutamyl transferase to the active agent 4-hydroxymethylphenylhydrazine (Figure 15.17), which is a well-known antipyridoxine factor. The hydrolysis of agaritine is accelerated if the cells of the mushrooms are disrupted. The mechanism underlying the antipyridoxine activity is believed to be condensation of the hydrazines with the carbonyl compounds pyridoxal and pyridoxal phosphate, resulting in the formation of inactive hydrazones.

HO

OH

H HO

Naematolin B OAc

H O

HO

O

H

Naematolone OAc H O

Figure 15.15

Chemical structures of naematolin B and naematolone found in Naematoloma fasciculare (Hypholoma fasciculare) mushroom.

HO

HO

N

Necatorin

O N

Necatorone

O

O

N

N OH OH

OH

N

N

N

O

O

O N

N

N

HO

HO

HO

HO

HO

HO

N

N

O

O N

N

N O N

OH

4,4'-Binecatorone Figure 15.16

10-Deoxy-4,4'-binecatorone

10,10'-Dideoxy-4,4'-binecatorone

Alkaloids isolated from Lactarius necator (ugly milk cap mushroom) having mutagenic activity similar to that of aflatoxin B1.


O HO2HC

NHNH C

(CH2)2

CH

COOH

NH2 Agaritine γ-Glutamyl transferase

O HO2HC

NHNH2

HO

C

(CH2)2

CH

COOH

NH2 4-(Hydroxymethyl)-phenylhydrazine Figure 15.17

Glutamic acid

Hydrolysis of agaritine, the antipyridoxine factor found in the commercially cultivated edible Agaricus bisporus mushroom.

15.11 CONCLUSIONS Although mushrooms do not compose a significant portion of the human diet, they are a major toxic hazard in terms of the rate of poisoning and the number of fatalities caused. There are, as yet, no infallible methods of distinguishing poisonous mushrooms from edible ones. Furthermore, their chemical composition appears to be quite dependent on several factors. They often seem to have variable effects on individuals. As a general rule, one should consume only personally known edible wild species. Eating unfamiliar species may be quite hazardous. With advances in food production technologies, a vast variety of edible mushroom species is now available year round for one’s enjoyment. Thus, there appears to be no need for mushroom gathering in the wild. However, despite the culinary enjoyment that mushrooms offer, the risks accompanying wild mushroom consumption are not commensurate with the benefits.

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Spoerke, D. G. and Rumack, B. H. 1992. General approach to mushroom poisoning. Recent Adv. Toxicol. Res. 3: 120–134. Stijve, T. 1978. Ethylidene gyromitrin and N-methyl-N-formylhydrazine in commercially available dried false morels, Gyromitra esculenta Fr. Mitt. Geb. Lebensm. Hyg. 69: 492–504. Stijve, T. 1979. Bufotenine concentrations in carpophores of Amanita citrina (Schff) S. F. Gray. Mitt. Geb. Lebensm. Hyg. 70:246–253. Stijve, T. and Kuyper, T. W. 1985. Occurrence of psilocybin in various higher fungi from several European countries. Planta Med. 385–387. Suortti, T. 1984. Improved analytical and preparative methods for necatorin from Lactarius necator (Fr.) Karst mushroom. J. Chromatogr. 301:303–307. Suortti, T. and von Wright, A. 1983. Isolation of a mutagenic fraction from aqueous extracts of the wild edible mushroom Lactarius necator. J. Chromatogr. 255:529–532. Takemoto, T., Nakajima, T., and Sakuma, R. 1964a. Isolation of an insecticidal constituent (ibotenic acid) from Amanita muscaria and A. pantherine. Yakugaku Zasshi 84: 1233–1234. Takemoto, T., Nakajima, T., and Yokobe, T. 1964b. Structure of ibotenic acid. Yakugaku Zasshi 84:1232–1233. Theobald, W., Buch, O., Kunz, H. A., Krupp, P., Stenger, E. G., and Heimann, H. Z. 1968. Pharmakologische und experimental-psychologische Untersuchungen mit 2 Inhaltsstoffen des Fliegenpilzes (Amanita muscaria). Arzneim. Forsch. 18:311–315.


Vergeer, P. P. 1983. Poisonous fungi: Mushrooms. In Fungi Pathogenic for Humans and Animals. Part B, eds. H.D.H. Howard and L. F. Howard, pp. 374–412. Marcel Dekker, New York. Von Clarmann, M. 1964. Pilzvergiftung. Fortschr. Med. 82: 508–529. Wieland, T. 1986. Peptides of poisonous Amanita mushrooms. In Springer Series in Molecular Biology, ed. A. Rich, pp. 256–271. Springer Verlag, New York. Wieland, T. and Faulstich, H. 1978. Amatoxins, phallotoxins, phallolysin, and antamanide: The biologically active components of poisonous Amanita mushrooms. CRC Crit. Rev. Biochem. 5:185–260. Wieland, T., Motzel, W., and Merz, H. 1953. Uber das Vorkommen von Bufotenin imgelben Knollenblatterpilz. Liebigs Ann. Chem. 577:10–16. Wieland, T. and Wieland, O. 1972. The toxic peptides of Amanita species. In Microbial Toxins, Vol. VIII, Fungal Toxins, eds. S. Kadis, A. Ciegler, and S. J. Ajl, pp. 212–239. Academic Press, New York. Winkelmann, M., Stangel, W., Schedel, I., and Grabensee, B. 1986. Severe hemolysis caused by antibodies against the mushroom Paxillus involutus and its therapy. Klin. Wochenschr. 64:935–938. Wisemann, J. S. and Abeles, R. H. 1979. Mechanism of inhibition of aldehyde dehydrogenase by cyclopropanone hydrate and the mushroom toxin coprine. Biochemistry 18:427–435. Zilker, T. 1987. Diagnose und Therapie der Pilzvergiftungen (Teil II). Leber, Magen, Darm 1987:173–197.

16 Toxic Metals, Radionuclides, and Food Packaging Contaminants

16.1 INTRODUCTION Incidental or unintentional food additives are substances present in our food that can alter its properties and have not been added deliberately. This group of food chemicals can be broadly classified under the general heading of contaminants and residues. These substances end up in our food supply by indirectly entering the food chain. Some of these chemicals are extremely hazardous to human health, whereas others are responsible for inducing plasmid-mediated drug resistance in several human pathogens. Still others may interact with natural food constituents, particularly the lipids, to generate free radicals with carcinogenic potency. Generally, most of these chemicals are broken down naturally or washed away. Nevertheless, residual amounts do remain in our food supply. Such unintentional addition is unfortunate but unavoidable. Incidental food additives have several sources: 1.

2.

3.

Contamination of soil and water supplies with heavy metals, radioisotopes, pesticides, and other toxic industrial chemicals Manufacturing processes, which may contribute packaging contaminants (monomers, polymer stabilizers, plasticizers, etc.), particles of the equipment used, and the remains of errant animals Chemicals applied to crops (plant hormones and pesticides, such as insecticides, fungicides, herbicides, and antisprouting agents) and administered to livestock and poultry to maintain or


improve their health (veterinary medicines, hormones, and feed additives) Any discussion of the hazards presented by the incidental contaminants must be cognizant of those features they may have in common. For example, although differing widely in chemical structures, they possess certain common physical properties that tend to increase their potential hazard to humans. Contaminants that are persistent in the environment resist degradation and are extremely stable. Second, they tend to accumulate in the human food chain, especially in fish, and it is this accumulation that renders them a potential hazard to humans. The characteristic that makes them an actual hazard is their slow rate of elimination and/or metabolism, which results in their accumulation in tissues. Finally, their toxicity is usually greater in higher-order mammals than in species of lower phylogenetic orders. For example, fish, seals, and crustaceans can tolerate much higher tissue levels of mercury and arsenic than can humans. Chemicals released into the environment that have one or more of these properties can be predicted to have a potential to cause human harm. Although not every environmental contaminant possesses all of these properties, there is a surprising similarity among these substances, to the extent that any aromatic halogenated substance or organometallic substance detected in the environment with these properties should be viewed with suspicion. In this chapter, the toxicological aspects of heavy metals, radioisotopes, and food packaging contaminants are described. Various toxicological aspects of pesticide

residues and industrial contaminants as well as drug residue contamination in our food chain are described in the two subsequent chapters.

16.2 TOXIC METALS Metals are considered the oldest toxins known to humans since the Stone Age. They are redistributed in the environment by natural geological and biological cycles. The biological cycles bioconcentrate elements through both plants and animals, through biomagnification in food cycles. Because of the elemental nature of metals and their diverse affinities for organic ligands in biological cycles, their toxicities arise as multiple organ effects more often than do those of any class of toxicants. In view of this multiplicity of effects, the concepts of critical organ and critical dose have evolved to delineate the most sensitive ones. Thus, the critical organ is the one showing adverse effects at the lowest dose; other organs and systems may be much more severely affected but only at higher doses. This concept is important with respect to toxicants for which a tolerance greater than zero has been assigned for technical or economic reasons (Nordberg, 1976; Ahmed, 1999). Factors that influence the toxicity of metals at a certain level of exposure are important, particularly in susceptible populations. These include metabolic interactions of essential metals, formation of metal-protein complexes, age and state of development of exposed individuals, life-style factors, chemical form or speciation, and immune status of the host (Burns et al., 1995; Fowler, 1991; Oehme, 1978). Toxicity of a metal is determined by the dose at the cellular level and such factors as chemical form (or speciation) and ligand binding (Ahmed, 1999). For example, alkyl compounds are lipid-soluble and pass readily across biological membranes, causing them to accumulate inside the cell, with the result that their toxicity differs from that of the inorganic form (e.g., alkylmercury is primarily neurotoxic, whereas mercuric chloride, HgCl2, is nephrotoxic). The strong attraction between metal ions and organic ligands influences not only their availability for absorption, but also the deposition of a metal in the body and its excretory route. Most biologically important metals bind strongly to tissues and therefore are slowly excreted, leading them to accumulate in vivo. Blood, urine, and hair are most frequently used to assess the exposure to metals and are thus often referred to as indicator tissues. Blood and urine exposure usually represent recent effects and correlate best with acute toxicity. Quantitation of metals in organs can be carried out by techniques such as atomic absorption, atomic fluorescence, flame emission, electron


probe, ion probe, neutron activation analysis, spark source mass spectroscopy, proton-induced x-ray emissions, x-ray fluorescence spectroscopy, and scanning x-ray analysis (Meloan, 1978; Jacobs, 1996; Ahmed, 1999). It is difficult to draw a clear distinction between essential—especially the trace elements in human nutrition—and toxic metals. Nearly all metals are toxic to humans if ingested in abnormal amounts. Similarly, the interaction of a toxic metal with an essential one occurs when they have similar metabolism. Moreover, absorption of a toxic metal from the gastrointestinal (GI) tract or lung is often influenced by an essential dietary element if they share—or if the toxic metal influences—a homeostatic mechanism, as for lead, cadmium, and iron. In addition, the physiological effects of some metals, such as cadmium, are closely related to the amount of other essential nutrients in the human diet (Reilly, 1991; Deshpande and Salunkhe, 1995). All metals are capable of interacting in the body with other cellular constituents. Nevertheless, it is possible to differentiate among elements that are known with certainty to be essential and those that display severe toxicological symptoms at extremely low levels and have no known beneficial physiological functions. The heavy metals, most noticeably mercury, lead, and cadmium, probably constitute the single largest group of elements that contaminate agricultural soils, water supplies, and the environment and eventually find their way into the human food chain. Other toxic metals include arsenic, beryllium, boron, selenium, and other metals and metalloids. The possible sources of contamination of our food and the physiologically adverse and toxic effects of these metals are described in the following section. 16.2.1

Sources of Contamination

Soil Soil is the primary source of toxic metals found in food crops. Although most nutrients are absorbed from the top 10–30 cm of soil, plants that are capable of developing extensive deep root systems can effectively penetrate the soils to a depth of more than 6–10 m. Hence, the toxic metal content of agricultural soils needs to be considered from the viewpoint of both surface contamination as well as the nature of the underlying soil and the surrounding area (Hall et al., 1953; Deshpande and Salunkhe, 1995). Although most toxic metal contamination of soils occurs because of environmental pollution, natural factors also play an important role in this regard. For example, the volcanic soils that are intensively cultivated in Java and Sumatra contain inherently high levels of mercury and other toxic metals. Reilly and associates (1989) reported the mercury and arsenic contents in soil, water, and foods

grown in the Dieng Plateau area of Java, where they found a significant accumulation of mercury in locally grown vegetables. It was estimated that the consumption of as little as 100 g potato each day would contain almost twice the World Health Organization’s (WHO) tolerable level of mercury intake. Accumulation by Pasture Plants and Crops Plants are capable of absorbing toxic metals from contaminated soils and accumulating them in various tissues. Selenium toxicity was first noticed in livestock grazing on pastures and herbage grown in selenium-rich soils (Knott and McCray, 1959; Gardiner et al., 1962). Cadmium poisoning of humans was reported in Japan when rice paddy fields were irrigated with water contaminated by effluent from a local zinc-cadmium-lead mine (Reilly, 1991). Similar incidences of cadmium, copper, and zinc toxicity were also reported in England and Zambia (Reilly and Reilly, 1971; Morgan, 1988). Some plants are also capable of absorbing and accumulating certain toxic metals in large amounts. The shrub Camellia sinesis absorbs large quantities of both aluminum and manganese from the soil and concentrates them in its leaves (Pennington, 1987). The dried leaves of this shrub are used to make tea. In the United Kingdom, tea drinking alone appears to contribute a significant dietary intake of manganese in the elderly population (Wenlock et al., 1979). Metal accumulation in foods thus is a natural occurrence, with benefits as well as possible disadvantages for those who consume the foods. Sewage Sludge The application of sewage sludge to agricultural lands constitutes a significant source of food contamination by toxic metals. Although it contains more than 40% organic matter and is a rich source of both nitrogen and phosphorus, sewage sludge, especially from heavily populated urban and industrial areas, can contain relatively high levels of several toxic metals. Incidences of crop failures from soils where such sludge is applied are not uncommon (Mackenzie and Purves, 1975). Normal concentrations of several metals in typical sludge samples are presented in Table 16.1. The metals are usually industrial in origin, although domestic waste also makes a substantial contribution. Mercury, zinc, lead, and cadmium have been reported to occur at high levels in household dust as well as in domestic garbage (Harrison, 1978; Price, 1988). The levels of toxic metals found in sewage sludge are considerably higher than those found in typical agricultural land, with more than 300 times as much zinc and 100 times as much boron and copper as


Table 16.1 Normal Ranges of Metals in Dry Matter of Sewage Sludge Metal Boron Cadmium Chromium Cobalt Copper Iron Lead Manganese Mercury Molybdenum Nickel Scandium Silver Titanium Vanadium Zinc

Content, mg/kg 15–1,000 60–1,500 40–8,800 2–260 200–8,000 6,000–62,000 120–3,000 150–2,500 3–77 2–30 20–5,300 2–15 5–150 1,000–4,500 20–400 700–49,000

Source: Compiled from Pike et al. (1975) and Capon (1981).

would occur in the normal arable rural soils (Berrow and Webber, 1972). Significant accumulation of these metals in food crops may result in potential health problems for the consumers. Of the metals found in sewage sludge, lead, cadmium, and mercury pose more serious problems than copper and zinc (Strenstrom and Vahter, 1974). Agricultural Chemicals and Fertilizers Some of the widely used commercial fertilizers are capable of introducing significant levels of cadmium to the soil. Such incidences have been reported from Sweden and Australia (Williams and David, 1973; Strenstrom and Vahter, 1974). The acid rain phenomenon observed in both Europe and North America further enhances the mobilization of toxic metals in agricultural soils, thereby facilitating a greater uptake by the food crops (Reilly, 1991). Certain toxic metals, e.g., mercury and arsenic, have also been used in both inorganic and organic forms in fungicides and other agrochemicals. The use of organomercurial compounds has been the cause of far more serious and better-documented cases of food poisoning in Iraq, Pakistan, and Guatemala (Bakir et al., 1973; Reilly, 1991). Mercurial compounds are no longer common in agricultural practices and have been largely replaced by less persistent fungicides. The use of arsenic pesticides in horticulture has similarly declined appreciably (MAFF, 1982).

Metal-Containing Water Contamination of both surface water and groundwater by industrial wastes is a prime source of heavy metal toxicity in human and animal nutrition. The contamination and subsequent consumption of seafood are particularly serious in this regard. The water used for food production and drinking, however, is often treated to remove excessive levels of toxic metals. Besides that by cadmium and mercury, large-scale pollution of water by other metals is quite common in several industrial countries (Prater, 1975; Reilly, 1991; Deshpande and Salunkhe, 1995). Food Processing Metal contamination may occur at several stages during food processing. Contamination sources include the following: 1. 2. 3. 4. 5.

The factory door Plant and equipment Catering operations Ceramic and enameled utensils Metal containers

Generally, only high-quality stainless steel, plastics, and other structural materials approved for contact with foods are used in food-processing plants. The use of ceramics and enameled utensils is a significant source of metal poisoning, particularly those made of aluminum, copper, lead, and cadmium, in the less-developed countries. Wrapping paper, cardboard containers, as well as the print and color applied to plastic containers are also capable of contaminating food (Klein et al., 1970; Watanabe, 1974; Heichel et al., 1974; Gramiccioni, 1984). 16.2.2

Occurrence and Toxicity

The occurrence and toxicity of some of the more common metal pollutants in the human food chain are briefly described in the following. Lead Of all the heavy metals, lead has probably the longest history of environmental contamination and toxicity to humans. Its presence in the human food chain continues to be a major health problem worldwide. Lead is the ubiquitous toxic metal and is detectable in practically all phases of the inert environment and in all biological systems. There is evidence that lead in the environment has increased during the past 200 years (Shukla and Leland, 1973). It is used on a very wide and increasing scale in the modern world, with production in the Western world alone totaling 4.8 million


tons per year (Deshpande and Salunkhe, 1995). For this reason, lead poisoning, or plumbism, has been intensely studied. Because lead is toxic to most living things at high exposure and there is no demonstrated biological need for it, a major issue is to determine the dose at which it becomes toxic. Specific toxicities vary with age and circumstances of the host, but the major risk is toxicity to the nervous system. The most susceptible populations are children, particularly toddlers, infants in the neonatal period, and fetuses (CDC, 1991; Ahmed, 1999). Lead is naturally present in the soil. Environmental lead is a product of storage batteries, ammunition, solder, pigment colors and dyes, galvanizing and plating processes, pipes, and insecticides. It is also used in alloys with antimony, tin, and copper. It is a constituent or a contaminant of houseware materials such as crystal and pewter. Tetraethyl lead is an antiknock additive in gasoline, introduced into the environment through exhaust fumes from leaded gasoline used in vehicles; in recent years, this use has been drastically curtailed worldwide. Because the diet, including drinking water, is considered the principal source of the total body burden of lead, lead contamination of foodstuffs and the possibility of chronic lead poisoning from this source are indeed important food toxicological problems. Estimates place the total contribution of dietary sources at 90% of the total body burden of lead (Concon, 1988). The daily intake of lead via food in human nutrition was estimated to be 100–300 µg, with considerably higher levels the result of increasing environmental pollution (Jelinek, 1992). At the international level, the United Nations (UN) Joint Food Agriculture Organization (FAO)/World Health Organization (WHO) Expert Committee on Food Additives (JECFA) expresses the tolerable intake of cumulative environmental contaminants having toxicological effects when taken in the diet on a weekly basis to allow for variations in intake levels. These values are expressed as provisional tolerable weekly intake (PTWI) (UNEP/FAO/ WHO, 1988). This committee recommended a PTWI for lead from all sources of 50 µg/kg body weight for adults. This value was based on the data collected from 26 countries in 1972 covering a wide variety of foods (WHO, 1972). Because of increased sensitivity of infants and children, JECFA lowered the PTWI for this group of the population to 25 µg/kg body weight in 1986. Lead content of some representative food groups is shown in Table 16.2. Generally, shellfish and finfish have higher lead content than milk, fruits, vegetables, and meat, and levels in mollusks and crustaceans are higher than in fish. Lead levels in kidney and liver are substantially higher than those in meat muscle.

Table 16.2 Lead Content of Selected Foods Food

Range, µg/100 g

Cereal grains Cereal grain products Seafood Raw Canned Meats Gelatin Eggs, whole Vegetables, leafy Legumes Raw, dried, or frozen Canned Apples Pear Milk Whole, fresh Skim, dried, and packaged Skim, bulk package Evaporated Tea, leaves Cocoa, dry Sugar, white Molasses Baking powder Yeast, dry Black pepper Cinnamon Nutmeg Allspice Chili powder Bay leaves Cider, apple Vinegar, cider Cola, 2 samples Ginger ale Beer, canned Wine, red Drinking water Alcoholic beverages

0–62 0–749 17–250 6–30 7–37 0–15 0–126 0–16 3–11

4–5

Mean 22 10.5 62 16 19 57 7 37 7 7 38 3 0 2 2 4.5 1.37 0.10

0–7

16–85 µg/L

1–50 µg/L 50–100 µg/L

53 150 117 40 11 41 64 v18 55 90 µg/L 100 µg/L 10 µg/L 40 µg/L 50 µg/L 5 µg/L

Source: Compiled from WHO (1976), Reilly (1991), Deshpande and Salunkhe (1995), and Janssen (1997).

The lead levels in vegetables are somewhat higher than in fruits, probably because of lead translocation from soil to the edible portion of vegetables compared with that of fruits grown on trees and bushes (Ahmed, 1999). Of all vegetables studied, spinach, with a large surface area compared to weight, usually contains high lead levels. Another factor affecting lead content of vegetation worthy of discussion is the growing location with respect


to major highways. Perhaps not surprisingly, there is a good correlation between average traffic counts and average soil and plant lead content at sites close to the roadside (Concon, 1988; Ahmed, 1999). As a corollary, an inverse relationship between distance from the road and lead content has been observed in various plants and vegetables. Processing and packaging also can significantly increase the total lead content in foodstuffs. In this regard, canning may markedly increase lead content. For example, in a study of 256 cans of food, the contents of 62% contained a lead level of 100 ppb or more, 37% contained 200 ppb or more, and 12% contained 400 ppb or more (Mitchell and Aldons, 1974). Of products in glass and aluminum containers, only 1% had lead levels in excess of 200 ppb. This is indicative of leaching of lead from the soldered seam of the can and is an especially serious problem with canned acidic foods, such as tomato paste. The consistently high lead content of canned foods unequivocally shows that canning and the attendant handling and processing operations significantly increase the degree of lead contamination of foodstuffs. Lead solder used in cans is a major controllable source of lead in food. Food processors in many countries have switched to nonsoldered cans. This is particularly important in the case of infant foods. The use of nonsoldered cans can cut lead concentrations on average to between one-fifth and one-tenth of previous levels. In contrast, attempts to decrease lead content by improving processing operations for food packed in lead-soldered cans have achieved only a 50% reduction (UNEP, 1992). However, nonsoldered cans are more expensive than lead-soldered cans and hence are not widely used. Drinking water consumed directly and used in food processing also contributes lead to the dietary intake. In addition to the efforts of FDA to control lead levels in foods, the EPA has also moved to limit lead intake from water and air. In 1993, WHO proposed a guideline value of 10 mg/L for lead in drinking water. The widespread use of lead piping and soldering of water tanks in some countries contributes to incidences of lead poisoning much higher than those attributable to leaded gasoline (Grobler et al., 1996). No organic forms of lead have been reported to occur in food. Lead in foodstuffs exists exclusively as salts, oxides, or sulfhydryl complexes. Most lead salts and oxides are insoluble in water, and, hence, lead absorption is low. The absorption of lead from food is estimated to be 10% in adults and 40% in children (Reilly, 1991). Several dietary factors influence the level of absorption. A low body-calcium status, iron deficiency, and diets rich in carbohydrates but lacking protein and those containing high levels of vitamin D result in increased absorption of lead.

In the normal adult, about 90% of the ingested lead is generally excreted in the urine and feces. The absorbed lead may be distributed into three compartments (Rabinowitz et al., 1973): (a) the freely diffusible lead, which probably includes blood lead and free exchangeable lead of soft tissues; (b) the more firmly bound but exchangeable soft tissue lead; and (c) the hard tissue lead, such as in bones, teeth, hair, and nails. Lead is present in practically every organ and tissue of the human body, with amounts ranging from 100 to 400 mg or about 1.7 µg/g tissue (Barry, 1975). Over 90% of the lead in the human body occurs in the bone. The retention of lead in soft tissues is greatest in liver, followed by kidneys, aorta, muscle, and brain in decreasing order (Whanger, 1982). Lead also passes the placental barrier readily. Blood and soft tissue lead are likely to be responsible for the symptoms of poisoning. Lead in the hard tissues is more tightly bound. However, an equilibrium state may exist among bone, blood, and soft tissue lead, so that hard tissue lead may be an important source of blood and soft tissue lead (Goyer and Chisolm, 1972). Lead accumulated in the hard tissues, therefore, must be viewed as potentially toxic. The levels of lead in the bones, teeth, and hair continue to increase with age, suggesting a gradual accumulation of lead in the body. Therefore, contamination of food with lead and the possibility of chronic lead intoxication through the diet necessitate constant monitoring. The halflife of lead in the hard tissues has been estimated to be greater than 20 years; that of blood lead is 27 days (Rabinowitz et al., 1973). The toxic effects of lead form a continuum from clinical or overt effects to subtle or biochemical effects, which involve several organ systems and biochemical activities. The symptoms of acute lead poisoning in humans are well documented. The major effects are related to hemopoietic, nervous, gastrointestinal, and renal functions (Reilly, 1991). Generally, anorexia, dyspepsia, and constipation are followed by an attack of colic with intense paroxysmal abdominal pain. Lead encephalopathy is also observed in young children (NAS, 1972; NRC, 1993). However, little is known about chronic lead poisoning over a long period. Mild anemia, mental deterioration, hyperkinetic or aggressive behavior, peripheral neuropathy, lead palsy, and kidney damage are some of the clinical symptoms of chronic lead poisoning (WHO, 1976). Kehoe (1966a) has listed four principles that govern the conditions under which lead poisoning may be induced: 1.

The time of appearance of symptoms of lead poisoning depends on the magnitude of the


2.

3. 4.

daily oral dose: i.e., large intakes cause symptoms to appear sooner. The dose, which is absorbed at a constant rate, is large enough that progressive accumulation of lead in critical quantities can occur within the lifetime of the individual. A critical concentration of lead must be attained in the blood. The likelihood of poisoning increases markedly if the concentration of lead in the tissues increases rapidly in response to relatively high dosage and there is a failure in some mechanism that inhibits the toxic effects of lead.

In Kehoe’s experiments with human volunteers, a dose of 0.62 mg lead daily was sufficient to bring about a slight accumulation of lead in the body; by extrapolation, it was estimated that daily dosages of 3.3, 2.3, and 1.3 mg produce a critical blood level of 80 mg/100 g in 8 months and 4 and 8 years, respectively. An oral intake of 10 to 15 mg of lead per day results in gastroenteric plumbism in about 30 days (Kehoe, 1966b). The toxic effects of lead and the minimal blood lead levels at which the effects are most likely observed are summarized in Table 16.3. Lead’s toxicological mode of action depends on its molecular configuration; inorganic lead, which is the form most available environmentally, contaminates foods, producing clinical signs different from those of organic forms (e.g., tetraethyl and tetramethyl lead) and is distributed differently in the body. Organic lead compounds may be absorbed in large quantities through the skin, but in these forms, their toxicities are primarily a problem of the petroleum industry (Ahmed, 1999). The organic forms are rapidly dealkylated by the liver to the trialkyl metabolites, which are responsible for toxicity. These metabolites in turn are slowly converted to inorganic lead (Hammond and Beliles, 1980). The most effective way to treat lead toxicity is removal of individuals from the source(s) of exposure. Chelation has a role in the treatment of symptomatic workers and children and is warranted in adults with blood lead levels above 60 µg/100 mL after assessment of biological and clinical parameters of exposure (Burns and Currie, 1995; Trachtenbarg, 1996). The Centers for Disease Control (CDC) in Atlanta has established guidelines to assist in evaluating exposure factors for lead toxicity in children (CDC, 1991). For children with severe lead poisoning, chelation is the standard procedure, even though the mortality rate may be 25%–35% when ethylene diaminetetraacetic acid (EDTA) or British AntiLewsite (BAL) is used individually; when

Table 16.3 Lowest Observed Effect Levels from Lead-Related Health Effects Blood lead concentration, µg/dL Effects Neurological Encephalopathy (overt) Hearing deficit Intelligence deficits In utero effects Peripheral neuropathy Hematological Anemia U-ALAa B-Eppb ALA inhibition Py-5-Nc inhibition Renal Neuropathy Vitamin D metabolism Blood pressure (males) Reproduction

Children

Adults

80–100 20 10–15 10–15 40

100–112

80–100 40 15 10 10

80–100 40 15 10

40

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