Foodborne Disease Handbook, Volume 3: Plant Toxicants 2nd Edition

Citation preview

Foodborne Disease Handbook Second Edition, Revised and Expanded Volume 3: Plant Toxicants

edited by

Y. H. Hui Science Technology System West Sacramento, California

R. A. Smith University of Kentucky Lexington, Kentucky

David G. Spoerke, Jr. Bristlecone Enterprises Denver, Colorado

MARCEL

MARCEL DEKKER, INC. D E K K E R

NEWYORK BASEL

ISBN: 0-8247-0343-X This book is printed on acid-free paper. Headquarters Marcel Dekker, Inc. 270 Madison Avenue, New York, NY 10016 tel: 2 12-696-9000; fax:2 12-685-4540 Eastern Hemisphere Distribution Marcel Dekker AG Hutgasse 4, Postfach 812, CH-4001 Basel, Switzerland tel: 41-61-261-8482; fax: 41-61-261-8896 World Wide Web http://www.dekker.conl The publisher offers discountson this book when ordered in bulk quantities. Formore information, write to Special Sales/Professional Marketing at the headquarters address above. Copyright 0 2001 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, by or any infornlation storage and retrieval system, without permission in writing from the publisher. Current printing (last digit): 1 0 9 8 7 6 5 4 3 3 1 PRINTED IN THE UNITED STATES OF AMERICA

Introduction to the Handbook

The Foodbome Disease Handbook, Second Edition,Revised am?Expanded, could not be appearing at a more auspicious time. Never before has the campaign for food safety been pursued so intensely on so many fronts in virtually every country around the world. This new edition reflects at least one of the many aspects of that intense and multifaceted campaign: namely, that research on food safety has been very productive in the years since the first edition appeared. The Handbook is now presented in four volumes instead of the three of the 1994 edition. The four volumes are composed of 86 chapters, a 22% increase over the 67 chapters of the first edition. Much of the information in the first edition has been carried forward to this new edition because that information is still as reliable and pertinent as it was in 1994. This integration of the older data with the latest research findings gives the reader a secure scientific foundation on which to base important decisions affecting the public's health. We are not so naive as to think that only scientific facts influence decisions affecting food safety. Political and economic factors and compelling national interests may carry greater weight in the minds of decision-makers than the scientific findings offered in this new edition. However, if persons in the higher levels of national governments and international agencies, such as the Codex Alimentarius Commission, the World Trade Organization, the World Health Organization, and the Food and Agriculture Organization, who must bear the burden of decision-making need and are willing to entertain scientific findings, then the infomation in these four volumes will serve them well indeed. During the last decade of the previous century, we witnessed an unprecedentedly intense and varied program of research on food safety, as we have already noted. There are compelling forces driving these research efforts. The traditional food-associated pathogens, parasites, and toxins of forty years ago still continue to cause problems today, and newer or less well-known species and strains present extraordinary challenges to human health. These newer threats may be serious even for the immunocompetent, but for the inlmunocompromised they can be devastating. The relative numbers of the immunocompromised in the world population are increasing daily. We include here not just those affected by the human immunodeficiency virus (HIV), but also the elderly; the very young; the recipients of radiation treatments, chemotherapy, and immunosuppressive drugs: iii

iv

to

Introduction

the Handbook

patients undergoing major invasive diagnostic or surgical procedures: and sufferers of debilitating diseases such as diabetes. To this daunting list of challenges must be added numerous instances of microbial resistance to antibiotics. Moreover, it is not yet clear how the great HACCP experiment will play out on the worldwide stage of food safety. Altruism and profit motivation have always made strange bedfellows in the food industry. It remains to be seen whether HACCP will succeed in wedding these two disparate motives into a unifying force for the benefit of all concerned-producers, manufacturers, retailers, and consumers. That HACCP shows great promise is thoroughly discussed in Volume 2, with an emphasis on sanitation in a public eating place. All the foregoing factors lend a sense of urgency to the task of rapidly identifying toxins, species, and strains of pathogens and parasites as etiologic agents, and of determining their roles in the epidemiology and epizootiology of disease outbreaks, which are described in detail throughout the Foodborne Disense Hmdbook. It is very fortunate for the consumer that there exists in the food industry a dedicated cadre of scientific specialists who scrutinize all aspects of food production and bring their expertise to bear on the potential hazards they know best. A good sampling of the kinds of work they do iscontained in these four new volumes of the Handbook. And the benefits of their research are obvious to the scientific specialist who wants to learn even more about food hazards, to the scientific generalist who is curious about everything and who will be delighted to find a good source of accurate, up-to-date information, and to consumers who care about what they eat. We are confident that these four volumes will provide competent, trustworthy, and timely information to inquiring readers, no matter what roles they may play in the global campaign to achieve food safety.

Y. H. Hui J. Richard Gorham Dcrvid Kitts K. D. Murre11 Wai-Kit Nip Merle D. Pierson Sved A. Suttar R. A. Smith David G. Spoerke, Jr. Peggv S. Stanjield

Preface

The world ofnature offers many pleasant attractions. Concurrent with theincreased crowding ofurban areas inmuchof the developed world, there is a growing tendency for stressed-out city dwellers to seek peace in the wilderness, the more or less easily accessible natural areas, both terrestrial and aquatic. Much of the fauna and flora of these natural areas are quite innocuous-for the most part, only specialists are aware of exceptions. And even some of the specialists might be unaware of hazards originating outside their own sphere of expertise. Among consumers, mushroom hunters and fishermen are probably the best informed about potential hazards in their favored haunts. However, without access to specialized equipment and laboratory protocols, even the most competent specialist may be quite as unable to detect a hazard in food as the most naive consumer. While poisonous mushrooms figure prominently in this volume of the Foodborne Diseuse Handbook, other dangerous botanicals are by no means neglected. By “dangerous,” we refer to a very broad range of effects on human and animal health. The poisonous plants, their toxins, and the symptoms they cause are all discussed in detail, but more than that, the reader will find current and helpful information on methods of chemical analysis and recommendations for the medical management of poisoning episodes. Mushrooms are enormously popular around the world as a food item. Fortunately for the average consumer, grocery stores and restaurants get their mushrooms from commercial growers. Such mushrooms have no inherent toxic properties and thus are considered safe to eat and, in fact, are safe to eat. However, even with commercially produced mushrooms, the potential for microbial and insecticidal contamination should not be ignored. In the category of organisms known as fungi, mushrooms and toadstools are relatively large and easy to recognize for what they are. There are other fungi, however, that most of us will never see and that many consumers do not even know exist. Yet they, or the toxins they elaborate, may be just as dangerous as the much more obvious poisonous mushrooms. These are the fungi that produce mycotoxins (e.g., aflatoxin). For example, edible plant foods may contain natural poisons. We have heard about molds in peanut, which are a form of fungi-and contain aflatoxin. Poisons in cotton seed, cabbage, and

V

vi

Preface

potatoes are usually either removed during processing or destroyed during cooking. Plant toxins are described in great detail-detection, identification, effects on human and animal health, epidemiology-in this volume.

Y. H. Hui R. A. Snzith David G. Spoerke, JK

Contents

htroduction to the Handbook Preface Corn-ibutors Colttents of Other Volumes

...

111

V

i.x xi

I. Poison Centers 1. U.S. Poison Centers for Exposures to Plant and Mushroom Toxins David G. Spoerke, Jr.

1

11. Selected Plant Toxicants 2. Toxicology of Naturally Occurring Chemicals in Food Ross C. Beier and Herbert N. Nigg

37

3. Poisonous Higher Plants Doreell Grace Lnng and R. A. Smith

187

4. Alkaloids R. A. Smith

247

5. Antinutritional Factors Related to Proteins and Amino Acids Irvin E. Liener

257

6. Glycosides Walter Majak and Michael H. Benn

299

vii

Contents

viii

7. Analytical Methodology for Plant Toxicants Alister David Muir

351

8. Medical Management and Plant Poisoning Robert H. Poppenga

413

9. Plant Toxicants and Livestock: Prevention and Management Michael H. RnIphs

44 1

111. Fungal Toxicants 10. Aspergillus ZoJin Kozakiewicz

47 1

11. Claviceps and Related Fungi Gretchen A. Kuldau and Charles W. Bacon

503

12. Fusarium Walter F. 0. Marasas

535

13. Penicillium John I. Pitt

581

14. Foodborne Disease and Mycotoxin Epidemiology S a m Hale Herwy and F. Xavier Bosch

593

15. Mycotoxicoses: The Effects of Interactions with Mycotoxins Heather A. Koshinshy, Adrienne Woytowich, and George G. Khnchntourians

627

16. Analytical Methodology for Mycotoxins James K. Porter

653

17. Mycotoxin Analysis: Immunological Techniques Fun S. Chu

683

18. Mushroom Biology: General Identification Features David G. Spoerke, Jr.

715

19. Identification of Mushroom Poisoning (Mycetismus), Epidemiology, and Medical Management David G. Spoerke, Jr.

20.

Index

Fungi in Folk Medicine and Society David G. Spoerke, Jr.

739

78 1

803

Contributors

Charles W. Bacon Toxicology and Mycotoxin Research Unit, Russell Research Center, Agricultural Research Service, U.S. Department of Agriculture, Athens, Georgia Ross C. Beier Southern Plains Agricultural Research Center, Agricultural Research Service, U.S. Department of Agriculture, College Station, Texas Michael H. Benn Chemistry Department, University of Calgary, Calgary, Alberta, Canada F. Xavier Bosch lona, Spain

Epidemiology Unit, Institute of Oncology, Llobregat Hospital, Barce-

Fun S. Chu Department of Food Microbiology and Toxicology, Food Research Institute, University of Wisconsin-Madison, Madison, Wisconsin Sara Hale Henry Center for Food Safety and Applied Nutrition, U.S. Food and Drug Administration, Washington, D.C. George G. Khachatourians Department of Applied Microbiology and Food Science, University of Saskatchewan, Saskatoon, Saskatchewan, Canada Heather A. Koshinsky Investigen, Alameda, California ZofiaKozakiewicz Biotechnology and Utilization of Biodiversity, CAB1 Bioscience, Egham, Surrey, England Gretchen A. Kuldau Department of Plant Pathology, Pennsylvania State University, University Park, Pennsylvania Doreen Grace Lang ington, Kentucky

Department of Veterinary Science, University of Kentucky, Lex-

Irvin E. Liener Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, St. Paul, Minnesota WalterMajak Range Research Unit, Agriculture and Agri-Food Canada, Kamloops, British Columbia, Canada ix

Contributors

X

Walter F. 0. Marasas Programme on Mycotoxins and Experimental Carcinogenesis, Medical Research Council, Tygerberg, South Africa Alister David Muir Crop Utilization, Agriculture and Agri-Food Canada, Saskatoon, Saskatchewan, Canada Herbert N. Nigg

University of Florida, Lake Alfred, Florida

John I. Pitt Food Science Australia, North Ryde, New South Wales, Australia Robert H. Poppenga New Bolton Center, School of Veterinary Medicine, University of Pennsylvania, Kennett Square, Pennsylvania James K. Porter Toxicology and Mycotoxin Research Unit, R. B. Russell Agricultural Research Center, Agricultural Research Service, U.S. Department of Agriculture, Athens, Georgia Michael H. Ralphs Poisonous Plant Research Lab, Agriculture Research Service, U.S. Department of Agriculture, Logan, Utah R.A.Smith Kentucky

Department of Veterinary Science, University of Kentucky, Lexington,

David G. Spoerke, Jr.

Bristlecone Enterprises, Denver, Colorado

Adrienne Woytowich Department of Applied Microbiology and Food Science, University of Saskatchewan, Saskatoon, Saskatchewan, Canada

Contents of Other Volumes

VOLUME 1: BACTERIAL PATHOGENS

I. Poison Centers 1.

The Role of U.S. Poison Centers in Bacterial Exposures David G. Spoerke, Jr.

11. Bacterial Pathogens 2. Bacterial Biota (Flora) in Foods James M. Jay 3. Aeromonns hydrophila Carlos Abeyta, Jr., Samuel A. Palumbo, and Gerard N. Stelma, Jr.

4. Update: Food Poisoning and Other Diseases Induced by Bacillus cereus Kathleerl T. Rnjkowski and James L. Smith 5.

Brucella Shirley M. Hallirzg and Edward J. Young

6. Campylobucter jejuni Don A. Franco and Charles E. Williams 7.

Clostridium botulirzum John W. Austift and Karerl L. Dodds

8.

Clostridium yel@irzgerzs Dorothy M. Wrigley xi

Contents of Other Volumes

xii

9. Escherichia coli Marguerite A. Neill, Phillip I. Tarr, David N. Taylor, and Marcia Wolf 10. Listeria nzonocytogenes Catherine W. Donnelly 11. Bacteriology of Salmonella Robin C. Arzderson and Richard L. Ziprirl

12. Salmonellosis in Animals David J. Nisbet and Richard L. Ziprin

13. Human Salmonellosis: General Medical Aspects Richard L. Ziprin and Michael H . Hume 14. Shigella Anthony T. Mnurelli crnd Keith A. Lnmpel

15. Stuphylococcus aureus Scott E. Martin, Eric R. Myers, and John J. Inndolo 16. Vibrio cholerae Charles A, Kaysner and June H. Wetherington 17. Vibrio yarahaernolyticus Tm-jyi Chai and John L. Pace

18. Vibrio vulnificus Anders Dalsgaard, Lise H@i, Debi Lirlkous, and James D. Oliver

19. Yersinia Scott A. Minnich, Michael J. Smith, Steven D. Weagant, and Peter Feng 111. Disease Surveillance, Investigation, and Indicator Organisms 20.

Surveillance of Foodborne Disease Ewen C. D. Todd

21. Investigating Foodborne Disease Dale L. Morse, Guthrie S. Birkhead, crnd Jack J. Guzewich

22. Indicator Organisms in Foods James M. Jay Index

Contents of Other Volumes

VOLUME 2: VIRUSES, PARASITES, PATHOGENS, AND HACCP I. Poison Centers 1. The Role of Poison Centers in the United States David G. Spoerke, Jr.

11. Viruses 2. Hepatitis A and E Viruses Theresa L. Cromeam, Michael 0. Favorov, Ornana V. Nainan, and Harold S. Margolis 3. Norwalk Virus and the Small Round Viruses Causing Foodborne Gastroenteritis Hazel Appleton

4. Rotavirus Syed A. Sattar, V. Susan Springthorpe, and Jason A. Tetro 5. Other Foodborne Viruses Syed A. Sattar and Jason A. Tetro

6. Detection of Human Enteric Viruses in Foods Lee-Ann Jaykus 7. Medical Management of Foodborne Viral Gastroenteritis and Hepatitis Suzanne M. Mntsui and Ramsey C. Chemg

8. Epidemiology of Foodborne Viral Infections Thomas M. Liithi 9. Environmental Considerations in Preventing the Foodborne Spread of Hepatitis A Syed A. Sattnr and Sabah Bidawid

111. Parasites 10. Taeniasis and Cysticercosis ZbignieMt S. Pawlowski and K. D. Murre11

11. Meatborne Helminth Infections: Trichinellosis William C. Campbell 12. Fish- and Invertebrate-Borne Helminths John H. Cross 13. Waterborne and Foodborne Protozoa Ronald Fayer

Contents of Other Volumes

xiv

14. Medical Management Paul Prociv

15. Immunodiagnosis of Infections with Cestodes Bruno Gottsteirr 16. Immunodiagnosis: Nematodes H. Ray Ganlble 17. Diagnosis of Toxoplasmosis Alan M. Johnson and J. P. Dubey 18. Seafood Parasites: Prevention, Inspection, and HACCP Arm M. A d a m and Debra D. DeVlieger

IV. HACCP and the Foodservice Industries 19. Foodservice Operations: HACCP Principles 0. Peter Snyder, Jr. 20. Foodservice Operations: HACCP Control Programs 0. Peter Srlyder, Jr. Irzdex

VOLUME 4: SEAFOOD AND ENVIRONMENTAL TOXINS I. Poison Centers 1. Seafood and Environmental Toxicant Exposures: The Role of Poison Centers Dmid G. Spoerke, Jr.

11. Seafood Toxins 2. Fish Toxins BrmP W. Hulstend

3. Other Poisonous Marine Animals Bruce W. Hdstend 4.

Shellfish Chemical Poisoning Ljwdolz E. Llewellyn

5. Pathogens Transmitted by Seafood Russell P. Herwig

Contents of Other Volumes

6. Laboratory Methodology for Shellfish Toxins David Kitts 7. Ciguatera Fish Poisoning Yoshitsugi Hokcrrna and Joanrle S. M. Yoshih-awa-Ebesu 8. Tetrodotoxin Joanne S. M. Yoshikawa-Ebesu, Yoshitsugi Hokanln, and Tarnno Noguchi

9. Epidemiology of Seafood Poisoning Lora E. Flemit1g, Dolores Kat:, Judv A. Bean, and Roberta Hammond 10. The Medical Management of Seafood Poisoning Donna Glad Blvthe, Eileerl Hack, Giavnnni Wnshington, and Lorn E. Fleming 11. The U.S. National Shellfish Sanitation Program Rebecca A. Reid m d Timothv D. Durance 12. HACCP, Seafood, and the U.S. Food and Drug Administration Kim R. Young, Miguel Rodrigues Kanznt, arrd George Perly Hoskin 111. Environmental Toxins

13. Toxicology and Risk Assessment Donuld J. Ecobichorr 14. Nutritional Toxicology David Kitts 15. Food Additives Laszlo P. Somogyi

16. Analysis of Aquatic Contaminants Joe W. Kiceniuk 17. Agricultural Chemicals Debra L. Browning and Carl K. Winter 18. Radioactivity in Food and Water Hank Kocol

19. Food Irradiation Hank Kocol 20. Drug Residues in Foods of Animal Origin Austin R. Long and Jose E. Roybnl

xv

xvi

Contents of Other Volumes

21. Migratory Chemicals from Food Containers and Preparation Utensils Yvonne V. Yuan 22. Food and Hard Foreign Objects: A Review J. Richard Gorhnm

23. Food, Filth, and Disease: A Review J. Richard Gorhnrn 24. Food Filth and Analytical Methodology: A Synopsis J. Richard Gorham

1 U S . Poison Centers for Exposures to Plant and Mushroom Toxins David G. Spoerke, Jr. Bristlecorte Enterprises, Denver, Colorado

Epidemiology I. A. B. C. D. E. F. G. H. 1. J. K.

1

AAPCC 2 Staffing poison a center 4 Types of calls received 5 How calls are handled 6 References used 7 How poisoncentersaremonitoredforquality Professionalandpubliceducationprograms Related toxicology organizations 8 International affiliations 10 Toxicology and poisoncenterWeb sites North American mycological association

11. PoisonInformationCenters

intheUnited

States

7

8

11 11 12

111. National and InternationalMycologicalAssociations/Clubs/ Organizations 23 References 36

1.

EPIDEMIOLOGY

Epidemiological studies aid treatment facilities in determining risk factors, determining who may become exposed, and establishing the probable outcomes of various treatments. A few organizations have attempted to gather such information and organize it into yearly reports. The American Associations of Poison Control Centers (AAPCC), North American Mycological Association (NAMA), and some federal agencies all work toward obtaining epidemiological information, while the AAPCC has an active role in assisting with the treatment of exposures. Epidemiological studies assist government and industry in determining package safety, effective treatment measures, conditions of exposure, and frequency of exposure. In 1987 there were 7023 cases of mushroom poisoning reported to the AAPCC. In 1988, that figure increased to 7,834 (1). These numbers were approxif

Spoerke

2

mately 0.6% of the total cases called to poison centers. The NAMA mushroom poisoning case registry was provided with 156 reports (4.6% of reported mushroom cases), and, in 1988, 116 cases (3.4%) were registered (1). Studies on mushroom poisonings provide information on the type of people most commonly involved in exposures. Are these patients children experimenting in the backyard, hikers, or mycophiles looking for dinner? Studies can also tell us which species are most commonly involved and what species were being sought. What types of symptoms are seen first, onset of symptoms, and any sequelae may also be determined and compared to accepted norms.

A.

AAPCC

1. What Are Poison Centers and the AAPCC? The group in the United States most concerned on a daily basis with poisonings due to household agents, industrial agents, and biologics (including plants and mushrooms) is the AAPCC. This is a national resource that provides information concerning all aspects of poisoning and often refers patients to treatment centers. This group of loosely affiliated centers is often supported by both government and industrial sources. Poison centers were started in the late 1950s, the first thought to be in the Chicago area. The idea caught on quickly and at the peak of the movement there were hundreds of centers throughout the United States. Unfortunately, there were little or no standards to define what might be called a poison center, the type of staff, hours of operation, or information resources. One center may have had a dedicated staff of doctors, pharmacists, and nurses trained specifically in handling poison cases: the next center may just have had a book on toxicology in the emergency room or hospital library. In 1993, the Health and Safety Code (Sec. 777.002) specified that a poison center provide a 24-hr service for public and health care professionals and meet requirements established by the AAPCC. This action helped the AAPCC to standardize activities and staffing of the various poison centers. The federal government does not fund poison centers, even though for every dollar spent on poison centers there is a savings of $2-9 in unnecessary expenses (2, 3). The federal agency responsible for the Poison Prevention Packaging Act is the U.S. Consumer Product Safety Commission (CPSC). The National Clearinghouse for Poison Control Centers initially collected data on poisonings and information on commercial product ingredients and biologic toxic agents. For several years the National Clearinghouse provided product and treatment information to the poison centers that handled the day-to-day management of the centers. At first most poison centers were funded by the hospital in which they were located. As the centers grew in size and number of calls being handled, both city and state governments took on the responsibility of contributing funds. In recent years, the local governments have found it very difficult to fund such operations and centers have had to look to private industry for additional funding. Government funding may take several forms, either as a line-item on a state’s budget, as a direct grant, or as moneys distributed on a per call basis. Some states with fewer residents may contract with a neighboring state to provide services to its residents. Some states are so populous that more than one center is funded by the state. Industrial funding also varies, sometimes as a grant, sometimes as

Poison Centers for Plant Toxin Exposure

3

Table 1 AAPCC MushroomExposures ~

~

~~~

~

Year

# of exposures

% accidental

% of total AAPCC calls

1989 1990

9388 9570

95 95

5.9 5.7

payment for handling the company’s poison or drug information-related calls, sometimes as payment for collection of data regarding exposure to the company’s product. Every year the AAPCC reports a summary of plant and mushroom exposures. As an example, data on mushroom exposures from 1989 and 1990 are listed in Tables 1 and 2. The totals do not equal loo%, as not everyone who was exposed to a mushroom went to anemergency department, and not all calls concerning mushroom exposures were due to poisonings. As can be seen by these statistics, there are a large number of exposures, but very few serious outcomes due to mushroom exposures. The same type of information is available for plant exposures. Each plant and mushroom has its own code number in the POISINDEX‘ reference system, which is entered by the poison center specialist taking the call. Thus, if the plant or mushroom is known at the time of exposure and the right code is entered, the database will describe ages, sexes, signs and symptoms, treatment, and outcomes for any particular plant or mushroom.

2. RegionalCenters The number of listed centers has dropped significantly since its peak of 600 plus. Many centers have been combined into regional centers. These regional poison centers provide poison information and telephone management and consultation, collect pertinent data, and deliver professional and public education. Cooperation between regional poison centersand poison treatment facilities is crucial. The regional poison information center should work with various hospitals to determine the capabilities of the treatment facilities of the region and to identify and have a working relationship with analytical toxicology laboratories, emergency departments, critical care wards, medical transportation systems, and extracorporeal elimination methods availability. This should be done for both adults and children. A “region” is usually determined by state authorities in conjunction with local health agencies and health care providers. Documentation of these state designations must be in writing unless a state chooses (in writing) not to designate any poison center or accepts a designation by other political or health jurisdictions. Regional poison infomation centers should serve a population base of greater than one million people and must receive at least 10,000 human exposure calls per year. The number of certified regional centers in the United States is now under SO. Certification as a regional center requires the following (4): Table 2 Outcomes of AAPCC Mushroom Exposures-% Outcomes Sx minor No or

Tx EDYear 1989 1990

24.4% 24.8%

78.5% 76.1%

of Total Calls: ED Visits and

Death Mod Sx Major Sx 2.5% 2.0%

0.16% 0.10%

3 cases 1 case

Spoerke

4

1. Maintenance of a 24 hr/day, 365 dayslyear service. 2. Service to both health care professionals and the public. 3. Availability of at least one specialist in poison information in the center at all times. 4. A medical director or qualified designee, on call by telephone, at all times. 5. Service readily accessible by telephone from all areas within the region. 6. Comprehensive poison information resources and comprehensive toxicology information covering both general and specific aspects of acute and chronic poisoning. 7. A list of on-call poison center specialty consultants. 8. Written operational guidelines, which provide a consistent approach to evaluation, follow-up, and management of toxic exposures. These guidelines must be approved in writing by the medical director of the program. 9. A staff of certified professionals manning the phones (at least one of the persons on the phone has to be a pharmacist or nurse with 2000 hr and 2000 cases of supervised experience). 10. A 24-hr/day physician (board certified) consultation service. 11. An ongoing quality assurance program. 12. Other criteria, as determined by the AAPCC, may be established with membership approval. 13. The regional poison information center must be an institutional member in good standing of the AAPCC. Many hospital emergency rooms still maintain a toxicology reference such as the POISINDEX system to handle routine exposure cases, but rely on regional poison centers to handle most of the calls in their area.

B. Staffing a PoisonCenter The staffing of a poison center varies considerably from center to center. The three professional groups most often involved are physicians, nurses, and pharmacists. Who answers the phones is somewhat dependent on the local labor pool, moneys available, and the types of calls being received. Other groups called on to serve in a center (with appropriate supervision) include students in medically related fields, toxicologists, and biologists. Persons responsible for answering the phones are either certified by the AAPCC or are in the process of obtaining the certification. Passage of an extensive examination in toxicology is required for initial certification, with periodic recertification required. Regardless of who takes the initial call, there is a medical director and other physician backup available. These physicians have specialized training or experience in toxicology, and are able to provide in-depth consultations for health care professionals calling a center. 1. Medical Director A poison center medical director should be board certified in medical toxicology, internal medicine, pediatrics, family medicine, or emergency medicine. The medical director should be able to demonstrate ongoing interest and expertise in toxicology as evidenced

PoisonPlant Centers for

Toxin Exposure

5

by publications, research, and meeting attendance. The medical director must have a medical staff appointment at a comprehensive poison treatment facility and must be involved in the management of poisoned patients.

2. ManagingDirector The managing director must be a registered nurse, pharmacist, or physician, or hold a degree in a health science discipline. The individual should be certified by the American Board of Medical Toxicology (for physicians) or by the American Board of Applied Toxicology (for nonphysicians). They must be able to demonstrate ongoing interest and expertise in toxicology. 3. Specialists in PoisonInformation These individuals must be registered nurses, pharmacists, or physicians, or be currently certified by the AAPCC as a specialist in poison information. Specialists in poison information must complete a training program approved by the medical director and must be certified by the AAPCC as a specialist in poison information within two examination administrations of their initial eligibility. Specialists not currently certified by the Associations must spend an annual average of no less than 16 hr/week in poison center related activities. Specialists currently certified by the AAPCC must spend an annual average of no less than 8 hr/week. Other poison information providers must have sufficient background to understand and interpret standard poison information resources and to transmit that information understandably to both health professionals and the public. 4. Consultants In addition to physicians specializing in toxicology, most centers also have lists of experts in many other fields. Poison center specialty consultants should be qualified by training or experience to provide sophisticated toxicology or patient care information in their area(s) of expertise. In regard to botanic exposures, the names and phone numbers of persons in a botanic garden, various nurseries, gardening clubs, or mushroom clubs are often available, with experts willing to donate their expertise in identification and handling cases within their specialty.

C. Types of Calls Received All types of calls are received by poison centers, most of which are handled immediately with a few others referred to more appropriate agencies. Which calls are referred depends on the center, its expertise, and the appropriateness of a referral. Below are types of calls that generally fall into each group. There is considerable variation between poison centers, and if there is doubt, call the poison center and they will tell you if your case is more appropriately referred. Poison centers do best on calls regarding acute exposures. Complicated calls regarding exposure to several agents over a long period that produce nonspecific symptoms are often referred to another medical specialist, to the toxicologist associated with the center, or to an appropriate government agency. The poison center will often follow up on these cases to track outcome and type of service given. Types of Calls Usually Accepted Drug identification. Actual acute exposure to a drug or chemical.

Spoerke

6

Actual acute exposure to a biologic agent (plants, mushrooms, various animals). Information regarding the toxic potential of an agent. Possible food poisoning. Drug information calls. Types of Calls Often Referred Questions regarding treatment of a medical condition (not poisoning). General psychiatric questions, with no drugs or chemicals involved. Proper disposal of household agents such as batteries, bleach, insecticides. Use of insecticides (e.g., which insecticide to use, how to use it) unless related to a health issue-for example, a person allergic to pyrethrins wanting to know which product does not contain pyrethrins). Drug information calls. 1. Data Collection AAPCC has certain rules about data collection. Records of all calls/cases handled by the center must be kept in a form that is acceptable as a medical record. The regional poison information center must submit all its human exposure data to the American Association of Poison Control Centers' National Data Collection System. The regional poison information center must tabulate its experience for regional program evaluation on at least an annual basis. In 1983 the AAPCC formed the AAPCC Toxic Exposure Surveillance System (TESS) from the former National Data Collection System. Currently TESS contains nearly 16.2 million human poison exposure cases. Sixty-five poison centers, representing 181.3 million people, participate in the data collection. The information has various uses to both governmental agencies and industry, providing data for product reformulations, repackaging, recall, bans, injury potential, and epidemiology. The summation of each year's surveillance is published in the Americcrn Jozmal of Emergency Medicine each year in late summer or fall (1 1, 12).

D. HowCallsAreHandled Most poison centers receive requests for information via the telephone. Calls come from both health care professionals and consumers. Only a few requests are received by mail or in person; these are often medicolegal or complex cases. Most centers can be reached by a toll-free phone number in the areas they serve, as well as a local number. Busy centers will have a single number that will ring on several lines. Calls are often direct referrals from the 91 1 system. In most cases, poison center specialists are unable to determine the exact plant or mushroom species, so it is difficult to give plant/mushroom-specific information. Often there is an attempt to at least identify the genus involved so as to estimate toxicity. In cases where few, if any, symptoms occur, and the more seriously toxic biologics can be ruled out, there is often minimal additional effort put forth to determine the plant/mushroom species. The patient is followed by telephone to assure no signs or symptoms develop. When symptoms are present, experienced plant/mushroom identifiers are often utilized to make identification as precise as possible withthe available botanic material. Poison information specialists listen to the caller, recording the history of the case on a standardized form developed by the AAPCC. Basic information such as the agent

Poison Centers for Plant Toxin Exposure

7

involved, the amount of agent, time of ingestion, symptoms, previous treatment, and current condition are recorded, as well as patient information such as sex, age, phone number, who is with the patient, relevant medical history, and sometimes patient address. All information is considered a part of a confidential medical record. The case is evaluated (using various references) as: 1. information only, no patient involved 2. harmless andnot requiring follow-up 3. slightly toxic, no treatment necessary but a follow-up call is given 4. potentially toxic, treattnent given at home and follow-up given until case resolution 5. potentially toxic, treatment may or may not be given at home, but it is necessary for the patient to be referred to a medical facility 6. emergency-an ambulance and/or paramedics are dispatched to the scene Cases are usually followed until symptoms have resolved. In cases where the patient is referred to a health care facility, the received agency is notified that the patient is in transit. The history is relayed, toxic potential discussed, and suggestions for treatment given. E. References Used References used also vary from center to center, but virtually all U.S. centers use a toxicology system called POISINDEX (5), which contains lists of products, their ingredients, and suggestions for treatment. The system is compiled using medical literature and medical specialists from throughout the world. Biologic products such as plants, insects, mushrooms, animal bites, and so forth are handled similarly. An entry for an individual plant might contain a description, toxic substance present, potential toxic amounts, and most dangerous plant part. The physician or poison information specialist is then referred to a treatment protocol that may be used for a general class of agents. An example would be: Pl?ilodend~-onexposures are referred to a protocol on oxalate-containing plants. This system is available on microfiche, a CD ROM, over a network, or on a mainframe. It is updated every 3 months. Not every plant and mushroom is on the system, but a great tnany are listed by both their scientific and common names. Various texts are also used. Among those mushroom sources stated as helpful in a survey of poison centers, Miller (6), Kingsbury (7), Rumack and Salzman (8), POISINDEX (5), Lincoff and Mitchel (9), and Stamets (10) are frequently mentioned. It is very difficult to identify plants and mushrooms using a description given over the phone, so often the assistance of garden club or mushroom club members, local greenhouses, botanic gardens, and various specialists is requested. Some poison centers have more experience with certain types of poisonings than do others, and these centers are often consulted during a more complex case. A recent trend has been for various manufacturers not to provide product information to all centers via POISINDEX, but to contract with one poison center to provide for poison information services for the whole country. Product information is given to only that center and cases throughout the country can only be handled by that one center.

F. How Poison Centers Are Monitored for Quality Most poison centers have a system of peer review. One person takes a call, another reviews it. Periodic spot review is done by supervisory and physician staff. General competence

Spoerke

8

is assured by certification and recertification via examination of physicians and poison informition specialists. Most regional centers have journal clubs where challenging cases are discussed.

G. ProfessionalandPublicEducationPrograms The regional poison information center is required to provide information on the management of poisoning to the health professionals throughout the region who care for poisoned patients. Public education programs, aimed at educating both children and adults about poisoning dangers and other necessary concepts related to poison control, should be provided. In the past, several centers provided stickers or logos such as Officer Ugh, Safety Sadie, and Mr. Yuck. These stickers could be placed on or near potentially toxic substances. While the intent was to identify potentially toxic substances that the children should keep away from, the practice has been much curtailed on the new assumption that in some cases the stickers actually attracted the children to the products. In the spring of every year there is a poison prevention week. National attention is focused on the problem of potentially toxic exposures. During this week many centers run special programs for the public. This may include lectures on prevention, potentially toxic agents in the home, potentially toxic biologic agents, or general first aid methods. Although this week is an important time for poison centers, public and professional education is a year-round commitment. Physicians often have medical toxicology rounds, journal clubs, and lectures by specialty consultants. Health fairs, school programs, and various women’s clubs are used to educate the public. The extent of these activities is often determined by the amount of funding from government, private organizations, and public donations.

H. RelatedProfessionalToxicologyOrganizations AACT American Association of Clinical Toxicologists Address: c/o Medical Toxicology Consultants; Four Columbia Drive; Suite 8 10; Tampa, FL 33606 AAPCC American Association of Poison Control Centers Address: 3201 New Mexico Avenue NW; Washington, DC 20016 Phone: 202-362-7217 FAX: 202-362-8377 ABAT American Board of Applied Toxicology Address: Truman Medical Center, West; 2301 Holmes St.; Kansas City, MO 64108 Phone: 8 16-556-3112 FAX: 816-881-6282 ABEM American Board of Emergency Medicine Address: 300 Coolidge Road; East Lansing, MI 48823 Phone: 5 17-332-4800 FAX: 517-332-2234 ACEP American College of Emergency Physicians (Toxicology Section) Address: P.O. Box 619911; Dallas, TX 75261-9911

Poison Centers forPlant Toxin Exposure

9

Phone: 800-798-1822 FAX: 214-580-2816 ACGIH American Conference of Governmental and Industrial Hygienists Address: Kemper Woods Center; Cincinnati, OH 45240 Phone: 5 13-742-2020 FAX: 513-742-3355 ACMT American College of Medical Toxicology (formerly ABMT) Address: 777 E. Park Drive; P.O. Box 8820; Harrisburg, PA 17105-8820. Phone: 717-558-7846 FAX: 7 17-558-7841 e-mail: [email protected] (Linda L. Koval) ACOEM American College of Occupational and Environmental Medicine Address: 55 West Seegers Road; Arlington Heights, IL 60005 Phone: 708-228-6850 FAX: 708-228- 1856 ACS Association of Clinical Scientists Address: Dept. of Laboratory Medicine; University of Connecticut Medical School; 263 Farmington Ave.; Farmington, CT 06030-2225 Phone: 203-679-2328 FAX: 203-679-2328 ACT American College of Toxicology Address: 9650 Rockville Pike; Bethesda, MD 20814 Phone: 30 1-571- 1840 FAX: 301-571-1852 AOEC Association of Occupational and Environmental Clinics Address: 1010 Vermont Ave., N W , #513; Washington, DC 20005 Phone: 202-347-4976 FAX: 202-347-4950 e-mail: [email protected] ASCEPT Australian Society of Clinical and Experimental Pharmacologists and Toxicologists Address: 145 Macquarie St.; Sydney N.S.W. 2000, Australia Phone: 6 1-2-256-5456 FAX: 6 1-2-252-3310 BTS British Toxicology Society Address: MJ Tucker, Zeneca Pharmaceuticals; 22B 11 Mareside; Alderley Park, Macclesfield; Cheshire, SKlO 4TG; United Kingdom Phone: 0428 65 5041 CAPCC Canadian Association of Poison Control Centers Address: Hopital Sainte-Justine; 3 175 Cote Sainte-Catherine; Montreal, Quebec H3TlC5 Phone: 5 14-345-4675 FAX: 5 14-345-4822 CSVVA (CEVAP) Center for the Study of Venoms and Venomous Animals Address: UNESP; Alameda Santos; N 647; CEP 01419-901; Sao Paulo, SP, Brazil Phone: 55 01 1 252 0233 FAX: 55 01 1 252 0200

Spoerke

10

EAPCCT European Association of Poison Control Centers Address: J. Vale; National Poisons Information Centre; P.O. Box 81898 d; N-0034 Oslo, Norway Phone: 47-260-8460 HPS Hungarian Pharmacological Society Address: Central Research Institute for Chemistry; Hungarian Academy of Sciences; H-1525 Budapest; P.O. Box 17; Pusztaszeri ut 59-67 Phone: 36-1-135-21 12 ISOMT International Society of Occupational Medicine and Toxicology Address: USC School of Medicine; 222 Oceanview Ave., Suite 100; Los Angeles, CA 90057 Phone: 213-365-4000 JSTS Japanese Society of Toxicological Sciences Address: Gakkai Center Building; 4-16, Yayoi 2-chome; Bunkyo-ku; Tokyo 113, Japan Phone: 3-38 12-3093 FAX: 3-38 12-3552 SOT Society of Toxicology Address: 1101 14th Street, Suite 1100; Washington, DC 20005-5601 Phone: 202-37 1- 1393 FAX: 202-371-1090 e-mail: [email protected] SOTC Society of Toxicology of Canada Address: P.O. Box 517; Beaconsfield, Quebec, H9W 5V1, Canada Phone: 5 14-428-2676 FAX: 5 14-482-8648 SSPT Swiss Society of Pharmacology and Toxicology Address: Peter Donatsch; Sandoz Pharma AG; Toxicologtie 88 1/130; CH4132 Muttenz, Switzerland Phone: 4 1-61-469-537 1 FAX: 4 1-61-469-6565 STP Society of Toxicologic Pathologists Address: 875 Kings Highway, Suite 200; Woodbury, NJ 08096-3172 Phone: 609-845-7220 FAX: 609-853-04 11 WFCT World Federation of Associations of Clinical Toxicology Centers and Poison Control Centers Address: Centre Anti-Poisons; Hopital Edonard Herriot; 5 pl d’Arsonva1; 69003 Lyon, France Phone: 33 72 54 80 22 FAX: 33 72 34 55 67 I. InternationalAffiliations AAPCC members attend various world conferences to learn of toxicology problems and new methods used by these agencies. An especially close relationship has formed between the American and Canadian Poison Center (CAPPC) associations. Once a year the AAPCC and CAPPC hold a joint scientific meeting and invite speakers and other toxicology spe-

Poison Centers for Plant Toxin Exposure

11

cialist from throughout the world to attend. Some international affiliated organizations are listed with the North American groups above.

J. Toxicology and Poison Center Web Sites Association of Occupational and Environmental Clinics This group is dedicated to higher standards of patient-centered, multidisciplinary care emphasizing prevention and total health through information sharing, quality service, and collaborative research. Address: [email protected]~~.edu Directory of Mycologists List of U.S. and Latin American mycologists who may be available for consultation. Address: http://www.keil.ukans.edu/-fungi Directory for MycorrhizalandEdibleFungi Address: http:// www.mykopat. slu.se/mycorrhiza/edible/home.pht1nl FingerLakesRegionalPoisonCenter Address: [email protected] Latin American Mycologists A site for identifying some Latin American mycologists. Address: http://bragg.ivic.velvic/ALM/directorio/direct/html Medical/Clinical/Occupational Toxicology Professional Groups A list of primarily U.S. professional groups interested in toxicology. There is a description of each group, its address, phone numbers, and contact names. Keyword: poison centers, toxicology. Address: http://www.pitt.edu/-martint/pages/motoxorg.htm Poison Net A mailing list dedicated to sharing information, problem solving, and networking in the areas of poisoning, poison control centers, hazardous materials, and related topics. The list is intended for health care professionals, not the lay public. The moderators do not encourage responses to individual poisoning cases from the public: Key word(s): poisoning, poison control centers K. NorthAmericanMycologicalAssociation In 1984 Ken Cochran started the Mushroom Poisoning Case Registry for the North American Mycological Association. This was kept at the University of Michigan until 1988, then was transferred toKen Lampe at the American Medical Association in Chicago. Since his death in 1990, John Trestrail TI1 has kept the records at the Blodgett Regional Poison Center in Grand Rapids, Michigan (1). Reports to the registry are made on a standard form, which is available free of charge or may be photocopied. The reporting is voluntary, and most often comes from physicians,

Table 3 NAMA MushroomExposures ~

~~

# of exposures

110

% nonhuman

# of genera

% unk. genera

6

26

15.5

Patient agea Under 6

6 13 to 12

10% ~~~

1%

to 17 0%

18 to 49

50 to 69

Over 70

37%

13%

6%

~~

These reports represent a different age distribution than seen in the AAPCC cases, where 80.5% are under 6 years of age.

Spoerke

12

poison centers, and mushroom club representatives. Because reporting is voluntary, it is also irregular. The number of cases recorded by NAMA is smaller than that reported by AAPCC, and they are not necessarily the same cases. Intepretation of these data and comparison to the AAPCC data is difficult, if not impossible. Other shortcomings of this database are that no species identifications are questioned, nor are any symptoms eliminated or evaluated based on the type of mushroom exposure. It is possible that symptoms reported could be due to coincidental illness, infection, or environmental conditions. Still, it is an attempt at gathering information on adverse reactions involving mushrooms. Table 3 is based on reports from 21 U.S. states or Canadian providences, but two ,states (Colorado and Oregon) represented 43% of the cases. This is thought to be due to more diligent reporting, rather than actual higher incidence of poisonings. These cases represent exposures reported in 1989 and 1990.

II. POISON INFORMATION CENTERS IN THE UNITED STATES The Poison Control Center telephone numbers and addresses listed below are thought to be accurate as of the date of publication. Poison Control Center telephone numbers or addresses may change. The address and phone number of the Poison Control Center nearest you should be frequently checked. If the number listed does not reach the poison center, contact the nearest emergency service, such as 9 11 or local hospital emergency rooms. The author disclaims any liability resulting from or relating to any inaccuracies or changes in the phone numbers provided below.

ALABAMA

ALASKA

Birmingham Regional Poison Control Center* Children’s Hospital of Alabama 1600 Seventh Avenue, South Birmingham, AL 35233-171 1 (800) 292-6678 (AL only) (205) 933-4050

Anchorage Anchorage Poison Center Providence Hospital P.O. Box 196604 3200 Providence Drive Anchorage, AK 99519-6604 (800) 478-3193 (AK only)

Tuscnloosa Alabama Poison Control System, Inc. 408 A Paul Bryant Drive, East Tuscaloosa, AL 35401 (800) 462-0800 (AL only) (205) 345-0600

Fairbanks Fairbanks Poison Center Fairbanks Memorial Hospital 1650 Cowles St. Fairbanks, AK 99701 (907) 456-7 182

* Indicates a Regional Center designated by the American Association of Poison Control Centers.

13

Poison Centers for Plant Toxin Exposure

ARIZONA Phoenix Samaritan Regional Poison Center* Good Samaritan Medical Center 1130 East McDowell Road, Suite A-5 Phoenix, AZ 85006 (602) 253-3334 Tucsorl Arizona Poison and Drug Information Center" Arizona Health Sciences Center, Room 1156 1501 N. Campbell Ave Tucson, A2 85724 (800) 362-0101 ( A Z only) (602) 626-6016

ARKANSAS Little Rock Arkansas Poison and Drug Information Center University of Arkansas College of Pharmacy 4301 West Markham, Slot 522 Little Rock, AR 77205 (800) 482-8948 (AR only) (501) 661-6161

CALIFORNIA Fresno Fresno Regional Poison Control Center* Fresno Community Hospital & Medical Center 2823 Fresno Street Fresno, CA 93721 (800) 346-5922 (CA only) (209) 445- 1222

Los Artgeles Los Angeles County University of Southern California Regional Poison Center* 1200 North State, Room 1107 Los Angeles, CA 90033

(800) 825-2722 (213) 222-2312 Orange University of California Irvine Medical Center Regional Poison Center" 101 The City Drive, South Route 78 Orange, CA 92668-3298 (800) 544-4404 (CA only) (714) 634-5988 Richmond Chevron Emergency Information Center 15299 San Pablo Avenue P.O. Box 4054 Richmond, CA 94804-0054 (800) 457-2202 (510) 233-3737 or 3738 Sacramento Regional Poison Control Center": University of California at Davis Medical Center 2315 Stockton Boulevard Rm HSF-124 Sacrmento, CA 95817 (800) 342-3293 (northern CA only) (916) 734-3692 Sun Diego San Diego Regional Poison Center* University of California at San Diego Medical Center 225 West Dickinson Street San Diego, CA 92013-8925 (800) 876-4766 (CA only) (619) 543-6000 Sun Francisco San Francisco Bay Area Poison Center* San Francisco General Hospital 1001 Potrero Avenue Rm 1E86 San Francisco, CA 94122 (800) 523-2222 (4 15) 476-6600 San Jose Regional Poison Center Santa Clara Valley Medical Center 751 South Bascom Avenue San Jose, CA 95 128 (800) 662-9886, 9887 (CA only) (408) 299-5112, 5113, 5114

Spoerke

14

COLORADO DerzverRocky Mountain Poison Center* 8802 Ninth Avenue Denver, CO 80220-6800 (800) 332-3073 (CO only) (303) 629-1123

CONNECTICUT Farnzington Connecticut Poison Control Center University of Connecticut Health Center 263 Farmington Avenue Farmington, CT 06030 (800) 343-2722 (CT only) (203) 679-3456

DELAWARE Wilmington Poison Information Center Medical Center of Delaware Wilmington Hospital 501 West 14th Street Wilmington, DE 19899 (302) 655-3389

DISTRICT OF COLUMBIA Wmhington National Capital Poison Center* Georgetown University Hospital 3800 Reservoir Road, North West Washington, DC 20007 (202) 625-3333

Tallahassee Tallahassee Memorial Regional Medical Center 1300 MiccosukEr Road Tallahassee, FL 32308 (904) 681-541 1 Tampa Tampa Poison Information Center* Tampa General Hospital Davis Islands P.O. Box 1289 Tampa, FL 33601 (800) 282-3171 (FL only) (813) 253-4444

GEORGIA Atlanta Georgia Regional Poison Control Center* Grady Memorial Hospital 80 Butler Street South East Box 26066 Atlanta, GA 30335-3801 (800) 282-5846 (GA only) (404) 6 16-9000

Macoil Regional Poison Control Center Medical Center of Central Georgia 777 Hemlock Street Macon, GA 31208 (912) 744-1146. 1100 or 1427 Sul~aililah Savannah Regional Poison Control Center Memorial Medical Center Inc. 4700 Waters Avenue Savannah, GA 31403 (912) 355-5228 or 356-5228

HAWAII FLORIDA Jacksonville Florida Poison Information Center University Medical Center 655 West Eighth Street Jacksonville, FL 32209 (904) 549-4465 or 764-7667

Honolulu Kapiolani Women's and Children's Medical Center 1319 Punahou Street Honolulu, HI 96826 (800) 362-3585, 3586 (HI only) (808) 941-4411

Poison Centers for Plant Toxin Exposure

IDAHO Boise Idaho Poison Center St. Alphonsus Regional Medical Center 1055 North Curtis Road Boise, ID 83706 (800) 632-8000 (ID only) (208) 378-2707

ILLINOIS Chicago Chicago and NE Tllinois Regional Poison Control Center Rush Presbyterian-St. Luke’s Medical Center 1653 West Congress Parkway Chicago, IL 60612 (800) 942-5969 (Northeast IL only) (312) 942-5969 Normal Bromenn Hospital Poison Center Virginia at Franklin Normal, IL 61761 (309) 454-6666

SprinRfield Central and Southern Illinois Poison Resource Center St. John’s Hospital 800 East Carpenter Street Springfield, IL 62769 (800) 252-2022 (IL only) (217) 753-3330 Urbancl National Animal Poison Control Center University of Illinois Department of Veterinary Biosciences 2001 South Lincoln Avenue, 1220 VMBSB Urbana, IL 61801 (800) 548-2423 (Subscribers only) (217) 333-2053

INDIANA Indianapolis Indiana Poison Center* Methodist Hospital

1701 North Senate Boulevard Indianapolis, IN 46202-1367 (800) 382-9097 (317) 929-2323

IOWA Des Moirles Variety Club Drug and Poison Information Center Iowa Methodist Medical Center 1200 Pleasant Street Des Moines, IA 50309 (800) 362-2327 ( 515 ) 24 1-6254

Iowa City University of Iowa Hospitals and Clinics 200 Hawkins Drive Iowa City, IA 52246 (800) 272-6477 or (800) 362-2327 (TA only) (319) 356-2922

Sioux C i v St. Luke’s Poison Center St. Luke’s Regional Medical Center 2720 Stone Park Boulevard Sioux City, IA 51 104 (800) 352-2222 (TA, NE, SD) (7 12) 277-2222

KANSAS Kunsas City Mid America Poison Center Kansas University Medical Center 39th and Rainbow Boulevard Room B-400 Kansas City, KS 66160-7231 (800) 332-6633 (KS only) (9 13) 588-6633 Topeka Stormont Vail Regional Medical Center Emergency Department 1500 West 10th Topeka, KS 66604 (9 13) 354-6100

Spoerke

16

Wichita Wesley Medical Center 550 North Hillside Avenue Wichita, KS 67214 (3 16)688-2222

Portland, ME 04102 (800) 442-6305 (ME only) (207) 87 1-2950

MARYLAND KENTUCKY St. Thomas Northern Kentucky Poison Information Center St. Luke Hospital 85 North Grand Avenue Ft. Thomas, KY 41075 (513) 872-5111

Louisville Kentucky Poison Control Center of Kosair Children’s Hospital 3 15 East Broadway P.O. Box 35070 Louisville, KY 40232 (800) 722-5725 (KY only) (502) 589-8222

LOUISIANA Houma Terrebonne General Medical Center Drug and Poison Information Center 936 East Main Street Houma, LA 70360 (504) 873-4069 Monroe Louisiana Drug and Poison Information Center Northeast Louisiana University School of Pharmacy, Sugar Hall Monroe. LA 7 1209-6430 (800) 256-9822 (LA only) (318) 362-5393

MAINE Portland Maine Poison Control Center Maine Medical Center 22 Bramhall Street

Baltimore Maryland Poison Center* University of Maryland School of Pharmacy 20 North Pine Street Baltimore, MD 21201 (800) 492-2414 (MD only) (410) 528-7701

MASSACHUSETTS Bostorl Massachusetts Poison Control System* The Children’s Hospital 300 Longwood Avenue Boston, MA 02115 (800) 682-9211 (MA only) (617) 232-2120 or 735-6607

MICHIGAN Adriarl Bixby Hospital Poison Center Emma L. Bixby Hospital 818 Riverside Avenue Adrian, MI 49221 (517) 263-2412 Detroit Poison Control Center Children’s Hospital of Michigan 3901 Beaubien Boulevard Detroit, MI 48201 Outside metropolitan Detroit; (800) 4626642 (MI only) (3 13) 745-57 1 1 Grand Rapids Blodgett Regional Poison Center 1840 Wealthy Street, South East Grand Rapids, MI 49506 Within MI: (800) 632-2727

Poison Centers for Plant Toxin Exposure Kalamazoo Bronson Poison Information Center 252 East Love11 Street Kalamazoo, MI 49007 (800) 442-4112 616 (MI only) (6 16)34 1-6409

MINNESOTA Minneapolis Hennepin Regional Poison Center” 701 Park Avenue South Minneapolis, MN 55415 (612) 347-3144 (612) 347-3141 (Petline) St. Paul Minnesotal Regional Poison Center* St. Paul-Ramsey Medical Center 640 Jackson Street St. Paul, MN 55101 (800) 222- 1222 (MN only) (612) 221-21 13

17 1465 South Grand Boulevard St. Louis, MO 63104 (800) 392-91 11 (MO only) (800) 366-8888 (MO, West IL) (314) 772-5200

MONTANA Denver Rocky Mountain Poison and Drug Center 645 Bannock St. Denver, CO 80204 (800) 525-5042 (MT only)

NEBRASKA Onmlza The Poison Center* Children’s Memorial Hospital 8301 Dodge Street Omaha, NE 681 14 (800) 955-9119 (WY, NE) (402) 390-5400, 5555

MISSISSIPPI Jackson University of Mississippi Medical Center 2500 North State Street Jackson, MS 39216 (601) 354-7660 Hattiesburg Forrest General Hospital 400 S. 28th Avenue Hattiesburg, MS 39402 (601) 288-4235

MISSOURI Kansas City Poison Control Center Children’s Mercy Hospital 2401 Gillham Road Kansas City, MO 64108-9898 (816) 234-3000 or 234-3430 St. Louis Regional Poison Center* Cardinal Glennon Children’s Hospital

NEVADA

L m Vegas Humana Hospital-Sunrise* 3 186Maryland Parkway Las Vegas, NV 89109 (800) 446-6179 (NV only) Reno Washoe Medical Center 77 Pringle Way Reno, NV 89520 (702) 328-4144

NEW HAMPSHIRE Lebanon New Hampshire Poison Center Dartmouth-Hitchcock Medical Center 1 Medical Center Drive Lebanon, NH 03756 (800) 562-8236 (NH only) (603) 650-5000

Spoerke

18

NEW JERSEY Newark New Jersey Poison Information and Education Systems* 201 Lyons Avenue Newark, NJ 07 112 (800) 962-1253 (NJ only) (201) 923-0764 Phillipsburg Warren Hospital Poison Control Center 185 Rosberg Street Phillipsburg, NJ 08865 (800) 962-1253 (908) 859-6768

NEW MEXICO Albuquerque New Mexico Poison and Drug Information Center” University of New Mexico Albuquerque, NM 87 131 (800) 432-6866 (NM only) (505) 843-255 1

NEW YORK BtrfSalo Western New York Poison Control Center Children’s Hospital of Buffalo 219 Bryant Street Buffalo, NY 14222 (800) 888-7655 (NY only) (716) 878-7654 Mineola Long Island Regional Poison Control Center* Winthrop University Hospital 259 First Street Mineola, NY 11501 (516) 542-2323, 2324, 2325 New York Cily New York City Poison Control Center* 455 First Avenue, Room 123 New York, NY 10016 (212) 340-4494 (2 12) 764-7667

Nyack Hudson Valley Regional Poison Center Nyack Hospital 160 North Midland Avenue Nyack. NY 10920 (800) 336-6997 (NY only) (914) 353-1000 Rochester Finger Lakes Regional Poison Control Center University of Rochester Medical Center 601 Elmwood Avenue Rochester, NY 14642 (800) 333-0543, (NY only) (716) 275-5151 Syracuse Central New York Poison Control Center SUNY Health Science Center 750 E Adams Street Syracuse, NY 13210 (800) 252-5655 (3 15) 476-4766

NORTH CAROLINA Asheville Western North Carolina Poison Control Center Memorial Mission Hospital 509 Biltmore Avenue Asheville, NC 28801 (800) 542-4225 (NC only) (704) 255-4490 or 258-9907 Charlotte Carolinas Poison Center Carolinas Medical Center 100 Blythe Boulevard Charlotte, NC 28232-2861 (800) 848-6946 (704) 355-4000 Durham Duke Regional Poison Control Center P.O. Box 3007 Durham, NC 27710 (800) 672-1697 (NC only) (919) 684-8111 Greensboro Triad Poison Center Moses H. Cone Memorial Hospital

19

Poison Centers for Plant Toxin Exposure 1200 North Elm Street Greensboro, NC 27401-1020 (800) 953-400 I (NC only) (919) 574-8105 Hickory Catawba Memorial Hospital Poison Control Center 810 Fairgrove Church Road, South East Hickory, NC 28602 (704) 322-6649

NORTH DAKOTA Fnrgo North Dakota Poison Center St. Luke’s Hospital 720 North 4th Street Fargo, ND 58122 (800) 732-2200 (ND only) (701 ) 234-5575

OHIO Akron Akron Regional Poison Center 281 Locust Street Akron, OH 44308 (800) 362-9922 (OH only) (216) 379-8562 Canto11 Stark County Poison Control Center Timken Mercy Medical Center 1320 Timken Mercy Drive, North West Canton, OH 44667 (800) 722-8662 (OH only) (216) 489-1304 Cincinnati South West Ohio Regional Poison Control System and Cincinnati Drug and Poison Infornlation Center* University of Cincinnati College of Medicine 231 Bethesda Avenue ML #144 Cincinnati, OH 45267-0144 (800) 872-51 11 (Southwest OH only) (513) 558-5111

Cleveland Greater Cleveland Poison Control Center 2074 Abington Road Cleveland, OH 44106 (216) 231-4455

Columbus Central Ohio Poison Center* 700 Children’s Drive Columbus, OH 43205 (800) 682-7625 (OH only) (6 14)228- 1323 Dayton West Ohio Regional Poison And Drug Information Center Children’s Medical Center One Children’s Plaza Dayton, OH 45404- 18 15 (800) 762-0727 (OH only) (5 13) 222-2227

Lorain County Poison Control Center Lorain Community Hospital 3700 Kolbe Road Lorain, OH 44053 (800) 821-8972 (OH only) (216) 282-2220 Snndusky

Firelands Community Hospital Poison Information Center 1101 Decatur Street Sandusky, OH 44870 (4 19) 626-7423

Toledo Poison Information Center of Northwest Ohio Medical College of Ohio Hospital 3000 Arlington Avenue Toledo, OH 49614 (800) 589-3897 (OH only) (419) 381-3897 YouilgstoM’n Mahoning Valley Poison Center St. Elizabeth Hospital Medical Center 1044 Belmont Avenue Youngstown, OH 44501 (800) 426-2348 (OH only) (216) 746-2222

Spoerke

20

Zclnesville Bethesda Poison Control Center Bethesda Hospital 2951 Maple Ave Zanesville, OH 43701 (800) 686-4221 (OH only) (6 14) 454-422 1

OKLAHOMA Oklahoma Cit?, Oklahoma Poison Control Center Children's Memorial Hospital 940 Northeast 13th Street Oklahoma City, OK 73104 (800) 522-4611 (OK only) (405) 27 1-5454

OREGON Portland Oregon Poison Center Oregon Health Sciences University 3181 South West Sam Jackson Park Road Portland, OR 97201 (800) 452-7165 (OR only) (503) 494-8968

PENNSYLVANIA Hershey Central Pennsylvania Poison Center* Milton Hershey Medical Center Pennsylvania State University P.O. Box 850 Hershey, PA 17033 (800) 521-61 10 (717) 531-6111 Lancclster Poison Control Center St. Joseph Hospital and Health Care Center 250 College Avenue Lancaster, PA 17604 (717) 299-4546 Philadelphia Philadelphia Poison Control Center* One Children's Center

34th and Civic Center Boulevard Philadelphia, PA 19104 (215) 386-2100 Pittsburgh Pittsburgh Poison Center" One Children's Place 3705 Fifth Avenue at DeSoto Street Pittsburgh, PA 15213 (412) 68 1-6669 Williclnlsport The Williamsport Hospital Poison Control Center 777 Rural Avenue Williamsport, PA 17701 (717) 321-2000

RHODE ISLAND Providence Rhode Island Poison Center" 593 Eddy Street Providence, RI 02903 (401) 444-5727

SOUTH CAROLINA Charlotte Carolinas Poison Center Carolinas Medical Center 1000 Blythe Boulevard Charlotte, NC 28232-2861 (800) 848-6946 Col~rmbia Palmetto Poison Center University of South Carolina College of Pharmacy Columbia, SC 29208 (800) 922-1 117 (SC only) (803) 765-7359

SOUTH DAKOTA Aberdeen Poison Control Center St. Luke's Midland Regional Medical Center 305 S. State Street Aberdeen, SD 57401

21

Poison Centers for Plant Toxin Exposure (800) 592-1889 (SD, MN, ND, WY) (605) 622-5678 Rapid City Rapid City Regional Poison Control Center 835 Fairmont Boulevard P.O. Box 6000 Rapid City, SD 57709 (605) 341-3333 Sioux Falls McKennan Poison Center McKennan Hospital 800 East 21st Street P.O. Box 5045 Sioux Falls, SD 571 17-5045 (800) 952-0123 (SD only) (800) 843-0505 (IA, MN, NE) (605) 336-3894

TENNESSEE Kno.uville Knoxville Poison Control Center University of Tennessee Memorial Research Center and Hospital 1924 Alcoa Highway Knoxville, TN 37920 (6 15) 544-9400 Memphis Southern Poison Center, Inc. Lebanheur Children’s Medical Center 848 Adams Avenue Memphis, TN 38103-2821 (901) 528-6048 Nushville Middle Tennessee Regional Poison Center, Inc . 501 Oxford House 1161 21st Avenue South B-101VUII Nashville, TN 37232-4632 (800) 288-9999 (TN only) (615) 322-6435

TEXAS

Medical Center Hospital 504 Medical Center Blvd. Conroe, TX 77304 (409) 539-7700 Drrllas North Central Texas Poison Center* Parkland Memorial Hospital 5201 Harry Hines Boulevard P.O. Box 35926 Dallas, TX 75235 (800) 441-0040 (TX only) (214) 590-5000 El Paso El Paso Poison Control Center Thomas General Hospital 4815 Alameda Avenue El Paso, TX 79905 (915) 533-1244 Galveston Texas State Poison Control Center University of Texas Medical Branch 8th and Mechanic Street Galveston, TX 77550-2780 (800) 392-8548 (TX only) (713) 654-1701 (Houston) (409) 765-1420 (Galveston) Lubbock Methodist Hospital Poison Control 3615 19th Street Lubbock, TX 79413 (806) 793-4366

UTAH Salt Like City Utah Poison Control Center* 410 Chipeta Way, Suite 230 Salt Lake City, UT 84108 (800) 456-7707 (UT only) (801) 581-2151

VERMONT

Conroe Burlington Vermont Poison Center Montgomery County Poison Information Center Hospital CenterMedical of Vermont

Spoerke

22 11 1 Colchester Avenue Burlington, VT 05401 (802) 658-3456

Parkersburg St. Joseph’s Hospital Center 19th Street and Murdoch Avenue Parkersburg, WV 26 101 (304) 424-4222

VIRGINIA Clzarlotteslille Blue Ridge Poison Center* University of Virginia Health Science Center Box 67 Charlottesville, VA 22901 (800) 45 1-1428 (VA only) (804) 924-5543

Richmond Virginia Poison Center Virginia Commonwealth University MCV Station Box 522 Richmond, VA 23298-0522 (800) 5526337 (VA only) (804) 786-9123

WASHINGTON Seattle Washington Poison Center 155 NE 100th Street, Suite #400 Seattle, WA 98105-8012 Within WA: (800) 732-6985 (206) 526-2121

WEST VIRGINIA Charleston West Virginia Poison Center* 31 10 MacCorkal Avenue S.E. Charleston, WV 25304 (304) 348-4211 (800) 642-3625 (WV only)

WISCONSIN Madison Regional Poison Control Center University of Wisconsin Hospital 600 Highland Avenue Madison, Wi 53792 (608) 262-3702 Milwaukee Poison Center of Eastern Wisconsin Children’s Hospital of Wisconsin 9000 West Wisconsin Avenue P.O. Box 1997 Milwaukee, WI 53201 (4 14) 266-2222

WYOMING Onraha. Nebraska The Poison Center c/o Mid-Plains Poison Center* Children’s Memorial Hospital 8301 Dodge Street Omaha, NE 68 114 (402) 390-5555 (800) 955-9119 (NE, ID, IA, KS, MO, SD)

Poison Centers for Plant Toxin Exposure

111.

23

NATIONAL AND INTERNATIONALMYCOLOGICAL ASSOCIATIONS/CLUBS/ORGANlZATlONS

ARGENTINA Asociacion Argentina de Micologia 1141 Parque San Francisco CP 5010 Cordoba, Argentina Instituto de Botanica C. Spegazzini Facultad de Ciencias Naturales y Museo de la Plata Calle 53 No 477 1900 La Plata, Buenos Aires, Argentina Universidad de Buenos Aires Laboratorio de Micologia Depart de Ciencias Biologicas Facultad de Ciencias Exactas y Naturales I1 Pabellon. 4 piso Ciudad Universitaria (Nunez) 1428 Buenos Aires, Argentina

Australian National Reference Laboratory in Medical Mycology The Royal North Shore Hospital of Sydney St. Leonards New South Wales 2065, Australia Plant Pathology Branch Herbarium New South Wales New South Wales Agriculture Biological and Chemical Research Institute Private Mailbag No. 10 Rydalmere New South Wales 2116, Australia Women’s and Children’s Hospital Mycology Laboratory Mycology Unit North Adelaide 5006, Australia

AUSTRIA ARMENIA Botanical Institute of the Academy of Sciences of Armenia 375063 Yerevan 63 Armenia

AUSTRALIA Australian Federation for Medical and Veterinary Mycology Mycology Laboratory Royal North Shore Hospital St Leonards New South Wales, 2065 Australia Australian Mushroom Growers Association PTY PO Box 265 Windsor, New South Wales, NSW 2756 Australia Australian Mycological Society Inc. Australian Biological Resources Study GPO Box 636 Canberra 2601, Australia

Osterreichische Mykologische Gesellschaft Institut fur Botanik Universitat Wien Rennweg 14 A-1300 Wein, Austria Verein fur Pilzkunde Tirol AchenseestrarSe 21 A 6200 Jenbach Tirol. Austria

BELGIUM Antwerpse Mykologische Kring Alfons Schneiderlaans 126 2100 Deurne Antwerpen, Belgium Institute of Hygiene and Epidemiology Mycology Section Rue Juliette Wytstnan 14 B-1050 Bruxelles, Belgium Mycotheque de L’Universite Catholique de Louvain Place Croix du Sud 3

Spoerke te6

B B-1348 Louvain-la-Neuve, Belgium National Garden of Belgium Domein van Bouchout B- 1860 Meise, Belgium

BRAZIL Instituto de Botanica Secao de Micologia i Liquenologia Caixa Postal 4.005 Sao Paulo, SP 01061-970 Brazil Unversidade Federal de Pernambuco Departmento de Micologia Centro de Ciencias Biologicas Cidade Universitaria 50670-420 Recife Pernambuco, Brazil

BRITAIN British Lichen Society Department of Botany The Natural History Museum Cromwell Road London, SW7 5BD, United Kingdom British Mycological Society PO Box 30 Stourbridge. West Midlands DY9 9PZ, United Kingdom International Mycological Institute Bakeham Lane Egham, Surrey TW209TY, United Kingdom

CANADA Alberta Department of Botany University of Toronto Toronto, Ontario, Canada M5S 1Al Edmonton Mycological Club 6003 109 B Ave Edmonton, Alberta, Canada T6A 1S7 University of Alberta Microfungus Collection and Herbarium Devonian Botanic Garden Edmonton, Alberta, Canada TG6 2E1 British CoIzmbin Department of Botany and Biology University of British Columbia Vancouver, BC, Canada V6T 2B1

Vancouver Mycological Society 403 Third Street New Westminster, BC V3L 2S1 Quebec Chibougamau Mycological Club 804 5e Rue Chibougamau, PQ, Canada G8P 1V4 Les Cercle des Mycologues de Montreal 4101 Rue Sherbrooke, Est. 125 Montreal, PQ, Canada HlX2B2 Le Cercle des Mycologues de Quebec Pavillon Comtois Universitaire de Lava1 Ste-Foy, PQ, Canada G1K 7P4 Les Cercle des Mycologues de Rimouski University of Quebec, Rimouski Rinlouski. PQ, Canada Les Cercle des Mycologues de Saguenay 438 Rue Perrault Chicoutimi, PQ, Canada G7J3Y9

Mycology Reference Laboratory Public Health Laboratory Myrtle Road Kingsdown, Bristol, BS2 8EL United Kingdom

Manitoba Department of Botany University of Manitoba Winnipeg, Manitoba, Canada

Royal Botanic Gardens The Herbarium Kew, Richmond Surrey, TW9 3AE, United Kingdom

Nova Scotia Acadia University Biology Department Wolfville, Nova Scotia, Canada BOP 1 x 0

25

Poison Centers for Plant Toxin Exposure Atlantic Regional Laboratory National Research Council 1411 Oxford Street Halifax, Nova Scotia Canada B3H3Z1 Ontario Canadian National Mycological Herbarium Centre for Land and Biological Resources Research William Saunders Building Agriculture Canada Ottawa, Ontario KIAOC6

Cultivated Mushroom Report University of Toronto Mississauga, Ontario Mycological Society of America Department of Botany University of Toronto, Erindale Campus Mississauga, Ontario, Canada L5LlC6 Mycological Society of Toronto 2 Deepwood Crescent North York, Ontario, Canada M3ClN8 Royal Ontario Museum Cryptogamic Herbarium c/o Department of Botany University of Toronto 25 Willcocks Street Toronto, Canada, M5S 3B2

CHINA (PEOPLE’S REPUBLIC) Academia Sinica Institute of Microbiology Zhong Guan Cun Hai Dian Beijing 100080 People’s Republic of China World Society for Mushroom Biology and Mushroom Products c/o Department of Biology The Chinese University of Hong Kong Shatin, New Territories, Hong Kong Mycological Society of the Republic of China Mycology Laboratory Department of Botany

National Taiwan University Roosevelt Road Section 4, No. 1 Taipei, Taiwan 10764. Republic of China Veterans General Hospital-Taipei Mycosis Research Laboratory Room 533, Medical Research Building c/o Departments of Pathology and Dermatology Shih-pai, Taipei City 11217 Taipei, Taiwan, Republic of China

COSTA RlCA University of Costa Rica School of Biology Escuela de Biologia Facultad de Ciencias Universidad de Costa Rica Cuidad Universitaria ‘Rodrigo Facio’ 2050 San Pedro de M. de Oca San Jose, Costa Rica

CUBA Asociacion Latinoamericana de Micologia Jardin Botanic0 Nacional Carretera del Rocio km 3.5 Calabazar, Boyeros CP 19230 Ciudad La Habana, Cuba

CZECH REPUBLIC Culture Collection of Basidiomycetes Laboratory of Biochemistry of WoodRotting Fungi Institute of Microbiology Academy of Sciences of the Czech Republic, Videnska 1083 142 20 Praha 4 Krc, Czech Republic Czech National Collection of Type Cultures National Institute of Public Health Srobarova 48 100 42 Praha 10 Czech Republic

Spoerke

26 Czech Scientific Society for Mycology PO Box 106 CZ-111 21 Praha 1 Czech Republic Itest Brozikova 451 Hradec Kralove 50012 Czech Republic

DENMARK Foreningen Til Svampekundskabens Fremme PO Box 102 DK-2860 Soborg, Denmark

ESTONIA Estonian Academy of Sciences Institute of Zoology and Botany/Tartu University Laboratory of Mycology 21 Vanemuise Street 202400 Tartu, Estonia Estonian Naturalists' Society Mycology Section Kompanii Street 3 EE2400 Tartu, Estonia

FINLAND Finnish Mycological Society Societas Mycologica Fennica Unioninkatu 44 SF-00170 Helsinki, Finland

FRANCE Association d'Ecologie et de Mycologie Laboratorie de Systematique et d'Ecologie Vegetale U.E.R. Pharmacie Rue Laguesse 59045 Little Cedex, France

Association Francaise de Lichenologie Laboratoire de Cryptogamie Universite Pierre et Marie Curie 7 quai Saint-Bernard 75230 Paris Cedex 05. France Museum National D'Historire Naturelle Laboratoire de Cryptogamie 12, rue Buffon 75005 Paris, France Observatoire Mycologique Neronde 7 1250 Mazille, France Societe Francaise de Mycologie Medicale Institut Pasteur 25 rue du Docteur Roux 75724 Paris, Cedex, France Societe Mycologique de France 18, rue de 1'Ermitage 75010 Paris, France

GERMANY Arbeitskreis Mykologie Deutsche Phytomedizinische Gesellschaft Technische Universitat Munchen Lehrstuhl fur Phytopathologie 85350 Freising-Weihenstephan, Gemany Bayerische Landesanstalt Fur Weinbau und Gartenbau Residenzplatz 3 D-97070 Wurzburg, Germany Botanischer Garten und Botanisches Museum Berlin-Dahlem Konigin-Luise Strass 6-8 D-14191 Berlin. Gernlany Deutsche Gesellschafr Fur Mykologie E.V. Rathausstrasse 16 D-78594 Gunnigen, Germany Deutschsprachige Mykologische Gesellschaft Mykologische Laboratorium Univ. Hautklinik Martinistrasse 52 D-2000 Hamburg 20, Germany

Poison Centers for Plant Toxin Exposure Gesellschaft Fur Natur und Umwelt Fachhaus Mykologie Abteilung Natur and Umwelt Postfach 34 1030 Berlin, Germany Institut fur Pflanzenschutz im Forst Biologische Bundesanstalt fur Land-und Forstwirtschaft Messeweg 11/ 12 38104 Braunschweig, Germany International Society for Human and Animal Mycology Brandelweg 24 D-793 12 Emmendingen, Germany International Society for Mushroom Science Institut fur Bodenbiologie Bundesfurschungsanstalt fur Landwirtschaft D-3300 Braunschweig Bundesalle 50, West Germany

GREECE University of Athens Culture Collections of Fungi Department of Biology Section of Ecology and Systematics Panepistinliopolis GR- 157 84 Athens, Greece

GUAM College of Agriculture and Life Sciences V 0 6 Station, Mangilao, Guam 96913 734-292 1x376

27

HUNGARY Hungarian Mycological Society Department of Botany University of Horticulture and Food Industry H- 118 Budapest Menesi ut 44, Hungary

ICELAND Akureyri Museum of Natural History PO Box 180 IS-602 Akureyri, Iceland

INDIA Banaras Hindu University Department of Mycology and Plant Pathology Herbarium Faculty of Agriculture Varanasi-221005, India Indian Mushroom Grower’s Association Indian Research Laboratory College of Agriculture Solon, Himachal Pradesh India International Journal of Research and Development National Centre for Mushroom Research and Training Chambaghat, Solan 173213 (HP) India Mycological Society of India Centre for Advanced Study in Botany University of Madras Madras 600 025. India

HONG KONG Mushroonl Journal of the Tropics The International Mushroom Society for the Tropics c/o Department of Botany Chinese University of Hong Kong Shatin, New Territories, Hong Kong

ITALY Associazione Micologica Ecologica Romana Piazza C. Finocchario Aprile 3 1-00081 Roma, Italy

Spoerke

28 La Rivista del Fungicoltore Modern0 40016 South Giorgio di Plano (BO) Postale Grupo IIII70 Bologna, Italy Universita Degli Studi Di Palermo Dipartimento di Scienze Botaniche Via Archirafi 38 1-90 123 Palmer0 Sicily, Italy

MEXICO College of Postgraduates Laboratory of Edible Mushroom Production Colegio de Postgraduados Apartado Postal 701 Puebla 72001 Puebla, Mexico Sociedad Mexicana de Micologia Apartado Postal 2-378 Mexico D.F. Mexico CP 02860, Mexico

JAPAN Japanese Association for Mycotoxicology Science University of Tokyo 12 Ichigaya Funagawara-Machi Shinjuku-Ku, Tokyo 162 Japan Mycological Society of Japan c/o Business Center for Academic Societies. Japan 4-16, Yayoi 2-chonle Bunkyo-ku, Tokyo 113, Japan Tottori Mycological Institute The Japan Kinoko Research Centre Foundation Kokoge 211, Tottori 689-11, Japan

KlRGHlZlSTAN National Academy of Sciences of Grghizistan Biological Institute Herbarium XXII Partesda Street 265 720071 Frunze, Kirghizistan

KOREA Korean Society of Mycology Department of Agrobiology College of Agriculture Dongguk University Seoul 100-715 Republic of Korea

NEPAL Department of Forests Forest Research and Infornlation Centre Babar Mahal PO Box 106 Kathmandu, Nepal

NETHERLANDS Centraalbureau Voor Schimmelcultures Oosterstraat 1 Post Office Box 273 3740 AG Baarn. Netherlands Centre for Soil Ecology: Biological Station Kampsweg 27 9418 PD Wijster, Netherlands International Association for Plant Taxonomy Nomenclature Cornnlittee for Fungi Centraalbureau voor Schimmelcultures PO Box 213 3740 AG Baarn, Netherlands Netherlands Mycological Society Nederlandse Mycologische Vereniging Biological Station Center for Soil Ecology Kampsweg 27 9418 PD Wijster, Netherlands Onderzoekinstituut RijksherbariuIdHortus Botanicus Department of Mycology PO Box 9514 2300 RA Leiden, Netherlands

29

Poison Centers for Plant Toxin Exposure

ZEALAND

NEW Victoria University Mycology Group School of Biological Sciences PO Box 600 Wellington, New Zealand

NORWAY Fungiflora AIS PO Box 95 Blindern, N-03 14 Research All-Russia Norway Oslo. Mycological Society of Fredrikstad Fredrikstad Soppforening PO Box 167 N-1601 Fredrikstad, Norway Norsk Soppforening PO Box 282 N- 1301 Sandvika, Norway

PHILIPPINES University of the Philippines at Los Banos Mycological Herbarium UPLB Museum of Natural History College, Laguna, Philippines

POLAND Polish Botanical Society Mycological Section Polskie Towarzystwo Botaniczne Aleje Ujazdowslue 4 00-478 Warszawa, Poland

All-Russia Plant Protection Institute Unit of Microbiological Plant Protection Podbelskogo Shosse 3 St. Petersburg-Pushkin 8 189620, Russia All-Russia Plant Protection Institute Jaczewski’s Mycology & Plant Phytopathology Laboratory Podbelskogo Shosse 3 St. Petersburg-Pushkin 8 189620, Russia Institute for Agricultural Microbiology Culture Collection of Microorganisms Podbelskogo Shosse 3 St. Petersburg-Pushkin 8 189620, Russia Komarov Botanical Institute Culture Collection of Basidiomycetes Russian Academy of Sciences Prof. Popov Street 2 Saint Petersburg, 197376 Russia Kornarov Botanical Institute Mycological and Lichenological Herbaria Russian Academy of Sciences Prof. Popov Street 2 Saint Petersburg, 197376 Russia Russian Botanical Society Conmission for the Investigation and Application of Mushrooms Komarov Botanical Institute Russian Academy of Sciences Prof. Popov Street 2 St. Petersburg 197376, Russia

SCOTLAND ROMANIA Societatea Micologica din Romania Aleea M. Sadoveanu Nr. 3 R-6600-Iasi-6, Romania

Botanical Society of Scotland Royal Botanic Garden Endinburgh, EH3 5LR, Scotland, United Kingdom

Spoerke

30 Royal Botanic Garden Edinburgh Inverleith Row Edinburgh. EH3 5LR, Scotland, United Kingdom

SINGAPORE National University of Singapore Botany Department Lower Kent Ridge Road Singapore 0511 Republic of Singapore

SOUTH AFRICA

,

National Collection of Fungi of the Republic of South Africa Mycology Unit Plant Protection Research Institute Private X134 Bag Pretoria 0001, Republic of South Africa South African Society for Plant Pathology Fruit and Fruit Technology Research Institute Private Bag X5013 Stellenbosch 7600 Republic of South Africa University of Pretoria Department of Botany Pretoria 0002. Republic of South Africa

SPAIN Asociacion Espanola de Espe.cialistas en Micolotia Servei de Microbiologia Clinica Hospital de Mar Passeig Maritim 25-29 08003 Barcelona, Spain Societat Catalana de Micologia Catedra de Botanica Facultat de Farmacia Universitat de Barcelona

Avenida Diagonal 643 08038 Barcelona Catalunya, Spain Universidad de Alcala de Henares Departamento de Biologie Vegetal (Seccion Mycologia) Facultad de Ciencias-28871 Madrid, Spain

SWEDEN Goteborg Mycology Club Goteborgs Svampklubb Halltorpsgatan 14 S-461 41 Trollhattan, Sweden Swedish Mycological Society Sveriges Mykologiska Forening Swedish Museum of Natural History PO Box 50 007 S-10405 Stockholm, Sweden University of Uppsala Botanical Museum Villavagen 6 S-752 36 Uppsala, Sweden

SWITZERLAND Swiss Mycological Society Societe Mycologique Suisse Institute de Botanique Universite de Nsuchatel Chantemerle 22, CH-2000 Neuchatel, Switzerland International Society for Human and Animal Mycology Gellerstrasse 11A CH-4052 Basel, Switzerland

THAILAND Chuylalongkorn University: Mushroom Research Unit Department of Botany Bangkok, 10330 Thailand

Poison Centers for Plant Toxin Exposure UNITED STATES National North American Mycological Society 4245 Redinger Rd Portsmouth, OH 45662

United States Federation for Culture Collections Roche Molecular Systems 1145 Atlantic Ave Alameda, CA US National Fungus Collections Systematic Botany and Mycology Laboratory USDA-Agricultural Research Service BOllA Room 304 10300 Baltimore Ave Beltsville, MD 20705-2350 Wadsworth Center for Laboratories and Research Laboratories for Mycology New York State Department of Health The Governor Nelson A. Rockefeller Empire State Plaza PO Box 509 Albany. NY 12201-0509 Alaska Alaska Mycological Society Box 2526 Homer, AK 99603 Glacier Bay Mycological Society PO Box 65 Gustavus, AK 99826-0065 Arkamus Arkansas Mycological Society 55 15 S Main St Pine Bluff, AR 71601-7452 Ccllifom icr Fungus Federation of Santa Cruz 1305 East Cliff Dr (Museum) Santa Cruz, CA 95062

Humboldt Bay Mycological Society PO Box 4419 Arcata, CA 95521-1419

31 Los Angeles Mycological Society Biology Department 5151 State University Dr Los Angeles, CA 90032 Mendocino County Mycological Society PO Box 87 Philo, CA 95466-0087 Mount Shasta Mycological Society 623 Pony Trail Mount Shasta, CA 96067 Mycological Society of San Francisco PO Box 11321 San Francisco, CA 94101-7321 Colorado Colorado Mycological Society PO Box 9621 Denver, CO 80209-0621 Denver Botanic Gardens Herbarium of Fungi 900 York St Denver, CO 80206 Pikes Peak Mycological Society PO Box 1961 Colorado Springs, CO80901- 196 1 Connecticut Connecticut Agricultural Experimental Station 123 Huntington St Box 1106 New Haven, CT 06504 Connecticut Valley Mycological Society 21 Johnson St Maugatuck, CT 06770 Nutmeg Mycological Society PO Box 530 Groton, CT 06340-0530 Georgia Centers for Disease Control Infectious Disease Section Atlanta, GA 30333 Southeastern Forest Experiment Station Forest Sciences Laboratory 320 Green St Athens, GA 30602-2044

Spoerke

32 Idaho Northern Idaho Mycological Association 5936 North Mount Carrol St Coeur d’Alene, ID 83814-9609 Southern Idaho Mycological Association PO Box 843 Boise, ID 83701 Illinois Agricultural Research Service Culture Collection Northern Regional Research Center 1815 North University St Peoria. IL 61604 Illinois Mycological Society 1183 Scott Ave Winnetka, IL 60093 International Mycological Association National Center for Agricultural Utilization Research 1815 North University St Peoria, IL 61604 Ioula Prairie States Mushroom Club 3 10 Central Dr Pella, IA 502 19- 190 1 Kansas Botany Department Department of Biology Pittsburgh State University Pittsburgh, KS 66762 Kaw Valley Mycological Society 601 Mississippi St Lawrence, KS 66044-2349 Department of Botany University of Kansas Lawrence. KS 66045 Kentrrch?! School of Biological Science University of Kentucky Lexington, KY 40506

Louisiana Gulf States Mycological Society 21 1 Lake Tahoe Dr Slidell. LA 70461-8536

Mavland American Type Culture Collection Mycology and Botany Department 12301 Parklawn Dr Rockville, MD 20852- 1776 Lower East Shore Mushroom Club RR 1, Box 94B Princess Anne, MD 21853-9711 Mycological Association of Washington 9408 Byeforde Rd Kensington, MD 20895-3606 Mycology Lab, USDA, ARS, NE Region Agr Research Center Beltsville, MD 20705 National Fungus Collections Plant Industry Station Beltsville, MD 20705 Massachusetts Berkshire Mycological Society Pleasant Valley Sanctuary Lenox, MA 02140 Boston Mycological Club 100 Memorial Dr Cambridge, MA 02142-1314 Farlow Reference Library and Herbarium of Cryptogamic Botany Harvard University 20 Divinity Ave Cambridge, MA 021 38 Michigan Blodgett Memorial Medical Center 1840 Wealthy. SE Grand Rapids, MI 49506 Department of Biology Central Michigan University Mt Pleasant, MI 48859 Michigan Mushroom Hunters Club 4255 19th St Wyandotte, MI 48192 239 Plant Biology Laboratory Michigan State University East Lansing, MI 48823

33

Poison Centers for Plant Toxin Exposure University Herbarium University of Michigan Ann Arbor, MI 48109 West Michigan Mycological Society 923 E Ludington Ave Ludington, MI 3943 1-2437 Minnesota Minnesota Mycological Society 7637 E River Rd Fridley, MN 55432-3058

304 Plant Pathology Building University of Minnesota St Paul, MN 55101 Shiitake News Forest Resource Center Rt. 2, Box 156A Lanesboro, MN 55949

Missouri Missouri Mycological Society Rural Route 3, Box 190 Concordia, MO 64020-9505 Nebraska American Bryological and Lichenological Society, Inc. Department of Biology University of Nebraska at Omaha Omaha. Nebraska 68 182-0072 New Hantpslzise Monadnock Mushroomers Unlimited PO Box 6296 Keene, NH 0343 1-6296

New Hampshire Mycological Society 84 Cannongate I11 Nashua, NH 03063-1948 New Jerse?) New Jersey Mycological Association 20 Lorraine Terr Boonton, NJ 07005

Maittake Inc. (Medicinal Mushrooms) PO Box 7634 6 Aster Ct Paramus, NJ 07653 New Mexico New Mexico Mycological Society 1511 Marble Ave N W Albuquerque, NM 87 104- 1347

New York Central New York Mycological Society 343 Randolph St Syracuse, NY 13205-2357

College of Forestry Syracuse University Syracuse, NY 13210 COMA RR 3, Box 137B Pound Ridge, NY 10576-9803 Come11 University Plant Pathology Herbarium Plant Science Building Cornel1 University Ithaca, NY 14853 Long Island Mycological Club PO Box 180081 Brooklyn, NY 11318 Mid-Hudson Mycological Association 43 South St Highland, NY 12528-9803 Mid-York Mycological Society 2995 Mohawk St Sauquoit, NY 13456 Mycologia Official Publication of the Mycological Society of America The New York Botanical Garden Bronx, NY 10458 Mycological Research Cambridge University Press North American Branch 40 West 20th St New York. NY 10011-4211 Mycotaxon PO Box 264 Ithaca, NY 14850 New York Mycological Society 140 W 13th St New York, NY 1001 1-7802 Rochester Area Mycological Society 71 1 Corwin Rd Rochester, NY 14610-2124 New York Botanical Gardens Bronx, NY 10458

Spoerke

34 North Cnrolinu Asheville Mushroom Club Nature Center, Gashes Center Road Asheville, NC 28805 Blue Ridge Mushroom Club PO Box 2032 North Wilkesboro, NY 28659-2032 Botany Library University of North Carolina 301 Coker, CB #3280 Chapel Hill, NC Cape Fear Mycological Society 10 Scots Hill Road Wilmington, NC 28405 Triangle Area Mushroom Club PO Box 61061 Durham, NC 3,7705 Ohio Ohio Mushroom Society 288 E North Ave East Palestine, OH 44413-2369 Oregon Department of Botany Oregon State University Corvallis, OR 97331 Eclectic Institute (Medicinal Mushrooms) 14385 S.E. Lusted Rd Sandy, OR 97055 Florence Mushroom Club Siltcoos Station Westlake, OR 97493 Lincoln County Mycological Society 207 Hudson Loop Toledo. OR 9739 1-9608 Mount Mazarna Mushroom Association 417 Garfield St Medford, OR 9750 1-4028

North American Truffling Society PO Box 296 Corvallis. OR 97339-0296 Oregon Coast Mycological Society PO Box 1590 Florence, OR 97439 Oregon Mycological Society 2781 S W Shenvood Dr Portland, OR 97201-2250 Willamette Valley Mushroom Society 2610 East Nob Hill Street SE Salem, OR 97302-4429 Pemsyhmia Dept of Biological Sciences Mellon Institute Carneige-Mellon University Pittsburgh, PA 15213

Mushroom News American Mushroom Institute 907 East Baltimore Pike Kennett Square, PA 193587

Rltocle Islmcl Mycological Society of America The Department of Botany University of Rhode Island Kingston. RI 02881 Temessee Department of Botany University of Tennessee Knoxville, TN 37916 Texus Association of Allergists for Mycological Investigations 444 Hermann Professional Building Houston, TX 77030

The Mushroom Grower's Newsletter c/o The Mushroom Company 464 Fulton St Klamath Falls, OR 97601

Medical Mycological Society of the Americas Department of Pathology University of Texas Health Service Center at San Antonio 7703 Floyd Curl Dr San Antonio, TX 78284-7750

M L ~ S ~ W OThe O ~ IJLo m w l Box 3156 Moscow, ID 83843

Texas Mycological Society 7445 Dillon Houston, TX 77061-2721

Poison Centers for Plant Toxin Exposure Utah Biology Dept, UMC53 Utah State University Logan, UT 84322 Vernlorlt Montshire Mycological Club RD No 1, Box 336 Windsor. VT 05089 Virginicc Department of Biology Virginia Polytechnic Institute and State University Blackburg, VA 24061 Wclshirlgton Fungi Perfecti P.O. Box 7634 Olympia, WA 98507 Kitsap Peninsula Mycological Society P.O. Box 265 Bremerton. WA 983 10-0054 Northwest Mushroomers Association 831 Mason St Bellingham, WA 98225 Olympic Mountain Mycological Society P.O. Box 270 Forks, WA 9833 1-0720 Pacific Northwest Key Council 124 Panorama Dr Chehalis, WA 98532-8628 Puget Sound Mycological Society University of Washington Urban Hort. GF- 15 Seattle, WA 98195-0001 Snohomish County Mycological Society P.O. Box 2822 Everett. WA South Sound Mushroom Club 6439 32nd Ave. NW Olympia, WA 98203-0822

35 Spokane Mushroom Club P.O. Box 2791 Spokane, WA 99220-2791 Tacoma Mushroom Society P.O. Box 99577 Tacoma. WA 98499-0577 Tri-Cities Mycological Society Rural Route 1, Box 5250 Richland, WA 99352 Twin Harbors Mushroom Club Route 2, Box 193 Hoquiam. WA 98550 Wenatchee Valley Mushroom Society 287 North Iowa Ave East Wenatchee, WA 98802-5205 Wisconsin Center for Forest Mycology Research Forest Products Laboratory USDA Forest Service 1 Gifford Pinchot Dr Madison. WI 53705 Section for Botany Milwaukee Public Museum 800 W Wells St Milwaukee. WI 53233 Northwestern Wisconsin Mycological Society Rural Route 03 Box 17 Frederic, WI 54837 Parkside Mycological Club 5219 85th St Kenosha, WI 53 142-4358 Wisconsin Mycological Society Room 614, MPM, 800 W Wells Milwaukee, WI 53233 Wyoming University of Wyoming Wilhelnl G. Solheim Mycological Herbarium Laramie, WY 82071

36

Spoerke

REFERENCES by certified regional poison 1. JH Trestrail, JLF Lampe. Mushroom toxicology resources utilized centers in the United States. Clin Toxicol 28:169-176, 1990. 2. CPSC. CPSC Chairman Ann Brown suggests information technology study to support work of poison centers. News Release #94-047, Tuesday, March 15, 1994. of regional poison con3. DL Harrison, JR Draugalis, MK Slack, PC Langly. Cost effectiveness trol centers. Arch Intern Med 156:2601-2608, 1996. 4. TG Martin. Summarizationof the American Associationof Poison Control Centers Certification Criteria for Regional Poison Information Centers. Internet Address: motoxorg.htm. 5. POISINDEXB Information System. Micronledex Inc. Englewood, CO, 1998. 6. OK Miller Jr. Mushrooms of North America. New York: EP Dutton, 1982. 7. JM Kingsbury. Poisonous Plants of the United States and Canada. Englewood Cliffs, NJ: Prentice-Hall,1964. 8. BH Rumack, E Salzman, eds. Mushroom Poisonings: Diagnosis and Treatment. West Palm Beach. FL: CRC Press, 1978. and Hallucinogenic Mushroom Poisoning. Dallas TX: Van 9. G Lincoff, DH Mitchel. Toxic Nostrand Reinhold, 1977. 10. P Stamets. Growing Gourmet and Medicinal Mushrooms. Berkeley, CA: Ten Speed Press, 1993. 11. TL Litovitz, BF Schmitz, KM Bailey. 1989 annual report of the American Association of Poison Centers National Data Collection System. Am J Emerg Med 8:394-442, 1990. 12. TL Litovitz, KM Bailey, BF Schmitz. KC Holm, W Klein-Schwartz. 1990 annual report of the American Association of Poison Centers National Data Collection System. Am J Emerg Med 9:461-509, 1991.

2 ToxicologJ of Naturally Occurring Chemicals in Food I

Ross C. Beier U.S. Department of Agriculture, College Station, Tesm

Herbert N. Nigg Univers-sihof Florida, Lake Aljjred, Florida

I. Introduction 39 A. Milk sickness 39 Phytoalexins B. 42 11. CyanogenicFoods46 A. General perspective 46 Cassava B. (Manihot) 48 111. Citrus 50

A. General perspective Limes B. 53

50

IV. Crucifers (Cruciferae,Brassica) A. Goitrogens 53 Carcinogenicity B. modulation V. Fruits and Vegetables(flavonoids) Dietary A. flavonoids 61 B. Biological effects

53

55 59

of flavonoids 64

67 VI. Herbs A. Asian medicinal herbs 68 B. Onion and garlic C. Yarrow 71 Herbal D. teas 71 E. Bay leaf 71

70

Mention of a trade name. proprietary product,or specific equipment doesnot constitute a guarantee or warranty by the U.S. Department of Agriculture and doesnot imply its approval to the exclusion of other products that may be suitable. This book chapter was prepared by a U.S. government employee as part of his official duties and legally cannot be copyrighted.

37

Beier and Njgg

38 F. G. H. I.

Bishop's weed seed 74 Rosemary and sage 74 Abortifacients 77 Psychoactive substances 77

VII. Mushrooms

77

A. Agaricus bisportls B. Gyromitra esculentn VIII. Mycotoxins A. B. C. D. E.

77 80

81

A Global perspective of food safety Food safety and public health hazard Ergot alkaloids in grain foods 86 Ergot alkaloids in cattle 88 Fumonisins 88

82 83

IX. Nightshades (Solanaceae) 91

A. B. C. D. E. F. G.

White potatoes 91 Cholinesterase inhibition 93 Glycoalkaloid content 93 Teratogens 97 Eggplant 98 Green peppers 99 Tomatoes 99

X. Nitrate-Rich Foods A. B. C. D. E.

100

Reduction of nitrate 100 Nitrosation of amines 100 Quantification of N-nitroso compounds Dietary nitrate intake 102 Nitrate levels in plants 104

101

XI. Parsleys (Uuzbellifeme) 105 A. Biological activities of linear furanocoumarins B. Celery 107 C. Parsley 110 D. Parsnips 112 E. Figs 112 XII. Oxalate-Rich Foods

105

113

A. General perspective 113 B. Mineral balance 114 C. Absorption 114 XIII.

Sweet Potatoes (Zpolnoen Baruras) 118

A. Proposed lung toxins 118 B. Average concentration ofthe lung toxin ipomeamarone C. Activation of the lung toxins 121 D. Sweet potato connection to high rates of asthma 122 XIV. Tannin-Rich Foods 122 A. General perspective B. Effects of tannins xv.

Conclusion

131

Acknowledgment References

137

137

122 123

120

Naturally Occurring Toxic Chemicals in Foods

1.

39

INTRODUCTION

The purpose of exploring the potential naturally occurring toxic hazards of food plants is not to suggest an irrational avoidance of these common foods. However, it is important to identify, define, and investigate the natural toxicants in our foods, provide some perspective on these chemicals, and show clearly that their toxicology is unknown in most cases. Many natural toxicants have functions similar to synthetic pesticides or other biohazardous chemicals. Humans apply synthetic pesticides to food and ornamental plants to prevent insect, fungal, and other pest damage. However, plants produce natural toxicants to protect themselves from pathogens and pests. The natural pesticide concentration in our foods may be as much as 10,000 times higher than that of synthetic pesticide residues (1). Because of the protection they provide to plants, these natural chemicals are prime candidates to be bred into plants by plant breeders and producers (2). The main consideration of the Committee on Food Protection, National Research Council, when reviewing natural toxicants in foods, was “the hope that it may contribute to a more informed, realistic, and sensible attitude on the part of the public toward the food supply” ( 3 ) . Natural toxic components in foods receive little study today, as was the case in 1966 (4). Most people routinely accept that plants eaten in their “pristine” state not only are absolutely safe for one’s health but are better than plants “manipulated” (e.g., pesticide treated or fertilized with manufactured nutrients) by humans. Many people believe that if thefood is natural, it naturally is good for you; however, consider these cases. The plant family Solanaceae includes species that are highly poisonous and also are used for some common medicinal drugs. For example, Solanurn nigl-urn L. and Atropa belltdonna L. are extracted for their bioactive drugs, including atropine, scopolamine, and hyoscyamine. Tobacco also is related to these plants, as are such common food plants as eggplants, garden peppers, tomatoes, and white potatoes. Livestock have died after ingesting potato vines, green potatoes, or tomato vines. Human poisoning episodes and fatalities also have been reported (5-10). There are a number of foods in the human food chain through which exposure to natural toxicants may occur. Some of these are meat, milk, eggs, fish, grains, fruits, herbs, vegetables, and liquids (beer, water, wine, etc.). The first example discussed here of the occurrence of a naturally occurring toxicant appearing in our food chain involves milk. This chapter then discusses notable examples of natural toxins in various food plants and contamination by mycotoxins.

A.

Milk Sickness

The disease in humans referred to as milk sickrzess, was first noted in North Carolina by the time of the American Revolution and today it still remains the classic example of tnilk poisoning. In animals, the disease is called frerrlbles, which is based on the signs of muscle trembling of poisoned animals. White snakeroot (Erqmtoriurn rugosum Houtt) is the etiological agent responsible for milk sickness in humans and trembles in animals, but over a century went by before the plant was connected with the disease. Milk sickness in humans was caused by the use of milk or milk products from animals consuming this plant. Trembles in animals was caused by directly ingesting the plant or, in young animals, by utilizing milk from poisoned mothers. A thorough description of the plant, habitat, historical aspects, and isolation of components are presented by Beier and Norman (1 1). White snakeroot may be found in damp open areas of the woods,

40

Beier and Nigg

shaded areas, along rivers, and in steep canyons. Figure 1 indicates the distribution of white snakeroot throughout the United States (12-18). The first written description of the milk sickness disease was in 1809 by Dr. Thomas Barbee (19). The first published name for the disease, sick stomach, was coined by an anonymous author in 1811. Nancy Hanks Lincoln was among those who died in 1818 during an epidemic at Pigeon Creek, Illinois, of the disease called milk sickness when her son, Abraham Lincoln, was 7 years old (20). Nancy’s great aunt and great uncle, as well as two neighbors, died within a few weeks of each other during the same epidemic. The disease progresses slowly in humans and is characterized by restlessness with vague pains, vomiting, loss of appetite, constipation, acetone breath, severe acidosis, coma, and death. Recovery from an attack is slow and may never be complete (21). The literature on white snakeroot is very vast as it has been written about since 1809. Unfortunately, information is so diverse and inconsistent that it is very difficult to follow the true story surrounding white snakeroot poisoning. It is interesting to note that a recent article by Molyneux and James incorrectly gives Drake credit for establishing the causal relationship for the disease (22). In fact, Drake’s outstanding reputation in the scientific community prompted the acceptance of his incorrect theory that poison ivy was the plant responsible for milk sickness, and stopped research into the real cause of the disease (19). White snakeroot poisoning is still a problem in horses and goats (1 1). In a single episode during the spring of 1985, 53 angora goats died of white snakeroot poisoning in central Texas, and the total loss at that ranch during the 1985 season was 85 goats (23). It was observed by ranchers and diagnostic laboratory personnel that the goats in the

Figure 1 Theshadedareaindicatesthedistribution States.

of white snakeroot throughout the United

Naturally Occurring Toxic Chemicals in Foods

41

central Texas area that had survived white snakeroot poisoning then apparently would leave the plant alone (J. C. Reagor, personal communications, 1987). The question of palatability of white snakeroot to the goat arises (24). The toxin in white snakeroot apparently is effective enough to prevent goats from eating the plant. Since goats are very sensitive to the white snakeroot toxin, they probably are using one of the mechanisms of learning in diet selection (25). The mechanism may be trial and error, since the toxin so quickly is devastating to the goat (J. C. Reagor, personal communications, 1987). The suspected causative agent in white snakeroot poisoning was tremetone (26,27). Three main ketones were isolated from white snakeroot: dehydrotremetone, tremetone, and hydroxytremetone (Fig. 2). However, synthetic tremetone was not toxic in animal tests (28), and again there was a lull in research on the cause of milk sickness. The availability of a good bioassay was the main stumbling block in determining the toxic components of white snakeroot. Many bioassays were evaluated for possible success in showing toxic activity to components from white snakeroot; a microsomal activation assay was selected (29). The assay allowed the isolation and identification of tremetone as the primary activatable toxic component in white snakeroot (Fig. 2) (30). Tremetone readily converts to dehydrotremetone in the plant and cell-free homogenates and also decomposes to dehydrotremetone in extracts. Dehydrotremetone is not toxic with or without microsomal activation. This efficient conversion and spontaneous decomposition of tremetone to dehydrotremetone explains why white snakeroot plant material and extracts have varied toxic activities (30). It also explains why Bowen et al. did not observe toxic activity with synthetic tremetone (28). Crude extracts of rayless goldenrod (Zsoconzn wrightii, jimmyweed) cause trembles and milk sickness in the southwestern United States

Tremetone

Hydroxytremetone

Oehydrotremetone Figure 2 Three main ketones isolated from white snakeroot. (From Ref. 26.)

Beier and Nigg

42

(31) and are positive in the microsomal activation assay, which may be due to tremetone (30). Other phytotoxins are commonly found in milk and meat. These are reviewed in Ref. 32. B. Phytoalexins Bell has reviewed ways in which plants may express resistance to pathogens (33). The production of phytoalexins (toxic chemicals) is a major mechanism of plant defense. There have been many definitions for the termphytonlexin; certainly, none seem to cover the complexity of biosynthesis or the range of biological activities of these compounds. A working definition of phytoalexins is “low molecular weight, antimicrobial compounds that are both synthesized by and accumulated in plants after exposure to microorganisms” (34, p. 734). Phytoalexins exhibit toxicity across much of the biological spectrum, and their activity is not confined just to microorganisms (35). Various chemical groups that comprise some of these phytoalexins are discussed in a number of reviews (36-38). These chemical groups include the coumarins, glycoalkaloids, isocoumarins, isoflavonoids, linear furanocoumarins, stilbenes, and terpenoids. An abbreviated list of phytoalexins found in some common food plants is presented in Table 1. Induction of phytoalexin synthesis can result from a plant’s exposure to many kinds of stimuli, for instance, bacterial or viral infection (39,40), exposure to cell-wall fragments Table 1 Phytoalexins in Some Food Plants

Plant Alfalfa Pea Soybean Bean Broadbean Grapes Cotton Peanut Celery Parsley Parsnip Rice Castor bean Potato Pepper Sweet potato Carrot Tomato Lima bean Tobacco Eggplant

Phytoalexins Vesitol, sativan, medicarpin Pisatin, cinnamylphenols, 2’-methoxychalcone Glyceollin Phaseolin Wyerone Viniferin, resveratrol Gossypol, cadalenes, lacinilenes, hemigossypol Resveratrol Furanocoumarins Furanocoumarins Furanocoumarins Momilactones, oryzalexins Casbene Rishitin, hydroxylubimin, phytuberin, a-solanine, a-chaconine, lubimin, solavetivone, phytuberol Capsidiol lpomeamarone 6-Methyoxymelleint falcarinol a-Tomatine, rishitin, falcarindiol, falcarinol 5-Deoxykievitol, 8,2’-dihydroxygenistein Rishltin, lubimin, phytuberin, phytuberol, solavetivone, capsidiol, glutinosone Lubimin

in Chemicals Toxic Occurring Naturally

Foods

43

(41-43), cold, ultraviolet (UV) light, heavy-metal salts (44), antibiotics, fungicides (38), herbicides (45), and at feeding sites of nematodes (46-48). Acidic fog can stimulate the phytoalexin response in celery (49). A single stimulus like the herbicide acifluorfen can increase the production of phytoalexins and stress metabolites in crops as diverse as bean, broad bean, celery, cotton, pea, pinto bean, soybean, and spinach (45). Since plants have the ability to increase the levels of phytoalexins in response to external stitnuli, it is important to know the foods in which an abundance of such natural chemicals tnay bea potential problem for humans. It also is important to understand how the chemical content of our foods can be altered unfavorably by various treatments during production, handling, processing, shipping, and marketing. In a hypothetical society that does not use synthetic pesticides, an environmental health scientist would be well advised to take a close look at the food consumed by the inhabitants of that society. Items for consideration include (50): 1. The number and types of naturally occurring compounds present in foods 2. The immense nutnber of chemically uncharacterized compounds present in foods 3. The unknown toxic effects of these chemicals 4. The level and frequency of human consumption of the compounds in foods These four reasons for scientific investigation of the food consumed in a hypothetical society do not differ from the real society in which we live. Thus, the chemical and toxicological study of food deserves serious attention. To help focus on naturally occurring pesticides as potential human toxicants, a Thanksgiving dinner menu developed by the American Council on Science and Health is presented in Table 2, and the potential toxicants are listed (5 1). The health effects of these toxicants include blood pressure elevation from tyramine (in wine) and antithyroid activity from glucosinolates (in broccoli). The dinner also includes a variety of such mutagens as eugenol (from cranberry sauce) and rodent carcinogens like the hydrazines (from mushrooms). In the 1978 review “Phytoalexins and Human Health,’’ phytoalexins of the garden pea (with pisatin), green bean (with phaseolin), and carrot (with chlorogenic acid and myristicin) (Fig. 3) were discussed (52). Carrot also contains the acetylenes carotatoxin (fruns-1,10-heptadecadiene-5,7-diyn-3-01)10 pg/g (53); falcarinol, 18.2 pg/g; falcarindiol, 41.6 pg/g; acetylfalcarindiol; and falcarinolone. Falcarindiol has antifungal activity and is possibly a phytoalexin (54). Carotatoxin is neurotoxic to mice with an LDso of about 100 pg/g and is toxic to Daphnin rnugna Straus (53). A number of food toxicants also are described in the books Food Toxicology, by J. M. Concon (55, 56). Theobromine (Fig. 4) in tea and cocoa powder (2%) may be a potential health hazard. Theobromine can produce testicular atrophy and spermatogenic cell abnormalities in rats (1). Achronic toxicity and Carcinogenicity study of cocoa powder in rats resulted in no carcinogenicity and only limited involvement of the heart, kidneys, and testes of rats at the highest levels of cocoa powder administered (57). Alfalfa sprouts fed to monkeys cause a severe syndrome sitnilar to lupus erythematosus (58). Crude cottonseed oil contains gossypol (Fig. 4) even when it is obtained from glandless cotton (59); this compound causes pathological changes in rat and human testes, and reversible sterility in males at an oral dose of about 10 tng/day (1). Gossypol is very toxic to swine and causes hydrothorax, hydropericardium, edema of the lungs, and hepatic necrosis (60). Hydrogenated oils used in margarine have a cis-trans isomerization of lipids that may

Beier and Nigg

44

Table 2 Thanksgiving Dinner Menu includescomposition Chemical

Course Appetizer Cream of mushroom soup Fresh vegetable tray Carrots Radishes Cherry tomatoes Celery Entree Roast turkey Bread stuffing with onions, celery, black pepper, mushrooms Cranberry sauce Choice of vegetable Lima beans Broccoli spears Baked potato Sweet potato Rolls with butter

Dessert Pumpkin pie with cinnamon and nutmeg Apple pie with cinnamon Beverages Coffee Tea Red wine Water Assorted nuts

Hydrazines Carotatoxin, myristicin, isoflavones, nitrate Glucosinolates, nitrate Hydrogen peroxide, nitrate, quercetin glycoside, tomatine Nitrate, psoralens Heterocyclic amines, malonaldehyde BenzoIaIpyrene, di- and trisulfides, ethyl carbamate, furan derivatives, hydrazines, psoralens, safrole Eugenol, furan derivatives Cyanogenic glycosides Allyl isothiocyanate, glucosinolates, goitrin,nitrate Amylase inhibitors, arsenic, chaconine, isoflavones, nitrate, oxalic acid, solanine Cyanogenic glycosides, furan derivatives, nitrate Amylase inhibitors, benzo[e]pyrene, ethyl carbamate, furan derivatives, diacetyl Myristicin, nitrate, safrole Acetaldehyde, isoflavones, phlorizin, quercetin glycosides, safrole Benzo(a]pyrene, caffeine, chlorogenic acid, hydrogen peroxide, methylglyoxal, tannins BenzoIaJpyrene, caffeine, quercetin glycosides, tannins Alcohol, ethyl carbamate, methylglyoxal, tannins, tyramine Nitrate Aflatoxins

Source: From Ref. 5 1.

play a role in cancer and aging. These are a few examples of the many naturally occurring potential toxicants in foods. As scientists, we have more than the simple responsibility of advancing the production rates and postharvest quality of our foods; we also are responsible for the wholesomeness and the subtle toxicological effects that foods may have on humans. It has been

Naturally Occurring Toxic Chemicals in Foods

45

HO

Phaseolin

Pisatin

OH

e

/

CH =CH H oHOo c a H OH

kH2CH =CH2

Myristicin

Chlorogenic acid Figure 3 Phytoalexinsfromvariousvegetables.

Theobromine 0

0

Gossypol Figure 4 Theobromine from tea and cocoa powder and gossypol from cotton-seed.

Beier and Nigg

46

estimated that as much as 35% of all cancer might be related to diet (61). Cancer is only one ailment that may be related to the presence of natural toxicants in our diet. There are many more subtle problems that potentially may be diet related, including the ubiquitous condition arthritis.

II. CYANOGENIC FOODS A.

GeneralPerspective

Cyanide wastes are a 5-6-billion-gallon problem in the United States (62) from industrial and food- and feed-production effluents (63). Cyanide poisoning is not uncommon (64, 65). Cyanide as hydrogen cyanide (HCN) is readily absorbed through skin, mucous membranes, and lungs (66). The American Conference of Governmental Industrial Hygienists threshold limit value (TLV) for exposure to cyanides is 10 mg/m3 and the TLV for hydrogen cyanide should not exceed 10 parts per million (ppm) or 11 mg/m3 (67). Skin is listed as a major route of exposure (67). Methacrylonitrile. with TLVs of 1 ppm or 2.7 mg/m3, is widely used in industry, and it is metabolized to hydrogen cyanide in mammals (67, 68). Hydrogen cyanide can also be a component in smoke and may lead to poisonings during fires (69-73). Cyanide poisoning from fire products may be confounded with or overshadowed by carbon monoxide poisoning (74-76); in rats, carbon monoxide and cyanide potentiate one another (77). The diagnosis of cyanide poisoning in fire victims is not unequivocal due to delays in analysis and possible postmortem cyanide production in blood (78) and direct cyanide diffusion (79). Newer, more sensitive analytical methods and more rapid postmortem analysis may clear up these unanswered questions (80). Many plants produce compounds that contain a cyano group and when eaten or crushed produce HCN, a process termed cymogenesis (81). For an excellent review of the biochemistry of selected cyanogenic glycosides, see Ref. 82. For reviews of cyanogenic compound occurrence, isolation, and characterization, see Refs. 83 and 84. Cyanide production requires p-glucosidases and other enzymes or acid conditions (81, 85-88). The pH of the human stomach (about pH 2) may not be low enough for this hydrolysis (81). Cyanogenic compounds usually are more toxic orally than by injection because the gut contains microorganisms with the necessary p-glucosidases to produce free HCN. p-Glucosidases also occur in various animal organs (89). Cyanogenic plants may be aproblem for livestock (90-92), but some range animals like sheep may adapt or tolerate consumption of these plant toxins (90,93,94). Mule deer consuming Saskatoon serviceberry (Arz-zelanchiernhifolia) went offtheir feed, lost muscle control, and died. Saskatoon serviceberry contains prunasin (95j. Adelzia volkensii, a cyanogenic perennial plant in Kenya, is used to kill hyenas and as a poison to commit suicide (96). In controlled experiments, Jackson administered 1.2, 0.7, and 0.4 mg/kg potassium cyanide orally to young swine (97). The following behavior changes were noted: fighting, decreased dominance behavior, vocalization, investigation of new environments, aggressive feeding, rooting, water overturning, increased distractibility from eating, anesthesia recovery, limping, limb stiffness, vomiting, and shivering. Poisonous plants present hazards to humans (98), but it is unexpected that food would present an acute or a chronic hazard. Around 1900 and also during World War I, lima beans imported into Europe from the tropics caused serious incidences of cyanide poisoning (99, 100). Lima beans implicated in fatal cases contained 200-300 mg/100 g or 2000-3000 yg/g of potential HCN. Lima beans that are acceptable for consumption

Naturally Occurring Toxic Chemicals in Foods

47

contain 100-200 pg/g of cyanogenic glycosides (100). Cyanide exposure has been linked to retrobulbar neuritis in pernicious anemia, tobacco amblyopia, subacute combined degeneration of the optic nerve in vitamin B I Zdeficiency, Leber’s hereditary optic nerve atrophy, and dominantly inherited optic nerve atrophy (101). These diseases apparently are linked to cyanide in tobacco smoke and to metabolic disorders in cyanide metabolism (101, 102). The lethal adult dose of liquid HCN is 50 mg; for cyanide salts it is 200-300 mg. The calculated lethal dosage in children is 1.2-5.0 mg/kg (pg/g) (71). The seeds of peaches, plums, cherries, bitter almonds, pears, apples, crab apples, apricots, pears, and cassavas all are cyanogenic. Canned unstoned peaches, apricots, plums, and morellos contained below 1.0 yg/g (ppm) of HCN in the pulp and syrup (1 03). Bamboo shoots may contain as much as 650 pg/g HCN potential ( 104). Sorghum, cassavas, peas, beans, and grams are cyanogenic (Table 3). Flax seed may contain up to 35,200 pg/g HCN potential.

Table 3 Cyanide Potential of Selected Foods (mg/kg)

Food

Pulp

Peach, fresh Peach, canned Plum, fresh Plum, canned Apricot, fresh Apricot, canned Morello, fresh Lima bean(Phaseolus

6.8 (amygdalin, av1.8 (prunasin) 103 erage of 4 varieties)

-

2.6

-

3.1 (averageof 8 varieties) 65

lunatus)

Fatalitiesa Normal (United States) Sorghum Cassava Linseed meal Black eyed pea (Vlgna

Reference No.

Seed

21 00-3120 140-1 67 2500 1130 530

0.12 9.8 1.5

103 103 103 103

0

103

29.5

103

0.5

-

-

99,100 99 99 99

21

99 99

sinensis)

Garden pea(Plsum satlvum) Kidney bean(Phaseolus vulgaris) Bengal gram(Cicer

23

99

20

99

0

99

arletinum) Red gram(Calanus calan)

5

99

29,000 2000 6000

85 85 85

Almond Bitter seed Young leaves Apricot, seed %lplicated in human fatalities.

Beier and Nigg

48

Cyanogenic glycosides in foodstuffs also may be acute poisons, sometimes resulting in death. Children poisoned from eating apricot seeds have been reported from Turkey (l05), where the apricot is a popular fruit and the seeds are processed and detoxified to produce tempe (106). Townsend and Boni reported a fatal apricot-ingestion case (107). A milk shake that included dried apricot kernels purchased at a health food store was the culprit. Humbert et al. (108) and Braico et al. (109) reported a child fatality from ingestion of one to five laetrile (amygdalin) tablets (Fig. 5). Sadoff et al. reported a laetrile death case in which a cancer patient taking the drug intravenously (IV) was unable to inject the drug and swallowed approximately 10 g instead (1 10). This 17-year-old girl died 24 hr later. Rubino and Davidoff reported a nonfatal poisoning case of a 49-year-old woman with nodular lymphoma who ate 20-40 apricot pits for lunch ( 1 1). Later analysis revealed that these pits contained 4090 pg/g of cyanide potential. Brian reported one death (a 7year-old girl) and one very ill 6-year-old girl after consumption of lima beans in New Guinea (1 12). A less-critical case of poisoning from cassava consumption also was reported ( 1 12). Stavric and Klassen studied the ability of fecal flora of the mouse, rat, hamster, guinea pig, monkey, and humans to hydrolyze amygdalin to HCN and benzaldehyde (1 13). Humans appeared to be the most-sensitive species because humans averaged 29% hydrolysis of the added amygdalin. One individual hydrolyzed 45%. Adults (35-55 years old) and children (3-6 years old) were about equal in this ability. Values for the other species were mouse (0.7%), guinea pig (2.2%), monkey (2.7%), rat (3.4%), and hamster (1 3%). Treated rats recovered quite quickly, resulting from a smaller rate of hydrolysis than humans. The oral LD25 forthe rat is 6.5 mg HCN/kg compared to the lethal oral dosage for humans of 0.5-1 .O mg HCN/kg (1 13).

B. Cassava (Manihof) Cassava (~nrzihot esculenta Crantz) is the major calorie source of an estimated 300 million people (1 14) and is the fourth most important food energy source in the tropics (1 15). It is native to tropical America and was used as food at least 4000 years ago (116). Cassava grows only in the tropics (it is cold sensitive), is bulky, high in energy and low in protein provision, and deteriorates rapidly after harvest (1 16, 117). It was projected to be used as food by 600 million people by the year 2000 (1 17). Cassava contains linamarin (Fig. 6) as the main cyanogenic glycoside, with smaller amounts of lotaustralin plus the enzyme linamarase, which liberates HCN from both com-

(R)-Amygdalin Figure 5 The cyanogenicdrug laetrile (amygdalin).

Naturally Occurring Toxic Chemicals in Foods

49

Linamarin Figure 6 Linamarin, the main cyanogenic glycoside in cassava (Mclnilzotesculerzta Crantz).

pounds (1 18). Cassava cultivars are classed as bitter or sweet. In general, bitter cultivars contain higher cyanogenic glycosides (1 18), but there is overlap between these classes and no correlation between taste and cyanogenic glycoside content has been made (1 18120). The cassava plant contains cyanogenic glycosides in all structures and content varies with cultivar, plant part, and growing conditions (1 18, 121, 122). Considerable research has been conducted on processing methods and their effect on cyanogenic glycoside content; for reviews, see Coursey (1 18) and Cooke and Coursey (1 16). Additional reviews include those on drying, wilting, chopping, and chemical treatments (124); fermentation (124, 125); flour source and fermentation (126); such traditional methods as blanching, crushing, and boiling (127, 128); cooking time and water temperature (129); extended processing and taste (fufu) (130); storage and water content (13 1); wheat/cassava flour bread (132); cassava variety floudwheat bread (133); and food analysis for cyanogenic glycosides (134). High-cassava diets are common in West Africa and an adult who consumes 750 g/ day may beexposed to 35 mg HCN, about one-half the lethal dose (135). The consumption of cassava is associated with goiter in iodine-deficient populations (136, 137). Cassava diets have been associated with goiter perhaps through thiocyanate (138, 139). Equivalent antithyroid activity in rats is produced by 10 g of cassava tubers and 1-2 mg SCN- (140). In Nigeria, a cassava diet also has been associated with ataxic neuropathy, myelopathy, bilateral optic atrophy, bilateral perceptive deafness, polyneuropathy (141), and death (1 42). Motor neuron disease, Parkinson's disease, cerebellar degeneration, psychosis, and dementia may accompany the disease and 35% of the patients displayed stomatoglossitis. The disease is linked to low sulfur intake, cassava cultivation, frequency of cassava meals, and plasma thiocyanate levels (141, 143, 144). In Zaire, symmetrical spastic paraparesis epidemics are associated with low sulfur amino acid intake and consumption of inadequately processed cassava (145). The disease, termed konzo, also has appeared in Mozambique (146), the United Republic of Tanzania (145, 147), and Liberia (148). Consumption of short-soaked cassava (one-day soaking) leads to the disease, whereas unaffected persons consumed cassava that had been soaked for 3 days during processing (149). Ankle clonus in children has been correlated with cassava intake and cyanide exposure in Mozambique (150). The consumption of food prepared from the cyanogenic cycad nut has been suggested as the cause of amyotrophic lateral sclerosis, Parkinson's disease, and dementia on Guam (15 1). However, cycads contain cycasins, which are carcinogens (152), mutagens, and neurotoxins. Metabolism of these compounds does not lead to HCN. For a review, see Ref. 153. Whole blood cyanide may be elevated in subjects, particularly those with sickle cell anemia, consuming cyanogenic plants (cassava) (154). Cyanide from smoking and from

Beier and Nigg

50

7-Hydroxy-6-methoxycoumarin Figure 7 Scopoletin (7-hydroxy-6-methoxycoumarin) in cassava may be the cause of optic atrophy and nerve deafness.

cassava consumption is postulated to cause calcific pancreatitis (155). Cruciferous plants contain l-cyano-2-hydroxy-3-butene,which, when administered by gavage to rats, resulted in modest elevation of urinary thiocyanate, but appeared to be a pancreatic toxin (156). Rhodanase, which converts cyanide to thiocyanate, is found in mammalian liver, lung, kidney, and brain (157). It is postulated that areas of the brain lacking rhodanase are targets for cyanide (157). Humans adapt to cyanide intake with increased conversion of cyanide to thiocyanate and production of antibodies (thiocyanate bound to a protein as an immunogen) (158). It is postulated that increased methionine and cysteine mobilization to provide sulfur for thiocyanate production and vitamin BI3utilization may operate as well (158). It has been suggested that eating cassava may be prophylactic for sickle cell anemia, bowel cancer, and schistosomiasis (159). A recent study suggests that optic nerve atrophy, nerve deafness, and endemic ataxia associated with cassava consumption may be due to scopoletin (7-hydroxy-6-methoxycoumarin) (Fig. 7), which also is found in cassava (160).

111.

CITRUS

A.

General Perspective

The total number of coumarins reported from natural sources was over 600 in 1977 (161), and that number increased by the year 1982 (162). Coumarins are naturally occurring chemicals that are distributed throughout the citrus species. Linear furanocoumarins are potent photosensitizing toxins (see Sec. XI) that also act as phytoalexins in citrus (163). Bergamot oil has been obtained from the peel of Citrus bergnmin for centuries and is used for its fragrant properties in perfumes. The use of a long-wavelength [320-400 nanometers (nm)] (UVA) sunscreen is more efficient for decreasing the phototoxic properties of bergamot oil than is a short wavelength (290-320 nm) (UVB) sunscreen (164). However, UVA and UVB sunscreens at the low concentrations found in perfumes cannot suppress the phototoxicity of bergamot oil on human skin. Citrus oils are pressed from the peel and used for flavoring candies, soft drinks, and baked goods. The coumarin content of cold-pressed lime oil is about 7% by weight, and that of orange oil is less than 0.5% (165). Solids recovered by column chromatography of cold-pressed citrus peel oils are shown in Table 4. These solids reflect the coumarin content of the citrus oils, except for bitter orange oil, which consists primarily of flavonoids. The coumarins and linear furanocoumarins present in citrus peel are nonvolatile and are not found in distillates, but, the best-quality oils are pressed directly from the peel without distillation.

Naturally Occurring Toxic Chemicals in Foods

51

Table 4 SolidsRecoveredfrom Cold-Pressed Citrus Peel Oils by Column Chromatography on Silicic Acid

Citrus oil

Weight Oo/

Lime, Mexican Grapefruit Bergamot Lemon Bitter orange

6.67 1.37 0.56

0.47 0.23

So~rrce:From Ref. 165.

In citrus juice, the main aromatic component is d-limonene (Fig. 8); it is present as 70-95% of the total volatile substances (166). Table 5 shows the d-limonene content of various orange juices and a grapefruit juice obtained by different techniques. These data led to the understanding that the retention behavior of citrus volatiles during the freezedrying process is based on physicochemical and biological characteristics of the food (166). The flavor of d-limonene was very dependent upon other nonvolatile, interfering constituents including acids, pectins, and sugars (167). There have been large improvements in the quantification of limonin, a compound that causes bitterness in juice processed from early-harvested citrus (168). More than 100 volatile flavor compounds (at very low concentrations) are found in citrus products. For a review, see Ref. 169. The quantitative analysis of the volatile constituents of lemon peel oil resulted in 51 constituents that accounted for approximately 99.7% of the total volatiles in both Sicilian and California commercial lemon peel oils (170). The three main components by weight of Sicilian lemon peel oil and California lemon peel oil, respectively, are limonene

FH3

&Limonene

O w o c H 3 6CH3

Lirnettin Figure 8 Components of citrus.

Beier and Nigg

52

Table 5 Concentrations of d-Limonene in Citrus Juices Obtained by Different Processes

Fruit Orange

Variety

Type"

squeezed Navel (2)b Fresh Orange Valencia Fresh squeezed Commercial Brands Brand A (2)b Orange Commercial pasteurized, single strength Brand B (3)' Orange Commercial frozen, reconstituted Grapefruit Brand Commercial reconstituted frozen,

53.8' 41.4 109.Ob 161.Oc 176.0

'The type of process used to produce the citrus juice. hNumber of observations of d-limonene content obtained from similarly processed citrus juice used to calculate the mean. cMean value. Source: From Ref. 166.

(70.43% and 70.53%), p-sinene (1 1.07%and 11.92%), and y-terpinene (1 0.03% and 8.89%). 7-Geranoxycoumarin is present in grapefruit, and isopimpinellin (Fig. 9) is present in lime oil, but neither occurs in lemon oil (165). Using such differences, reliable methods have been developed for detecting cross contamination of these citrus oils (171). With few exceptions, the compounds found in the citrus family are derived from psoralen (Fig. 9) and coumarin (165). The linear furanocoumarins found in citrus are psoralen (172), bergaptol, bergapten (Fig. 9), and bergamottin in grapefruit: phellopterin, 8-geranoxypsoralen, and bergamottin in lemon; and bergaptol and bergapten in orange (173). For an extensive review of the chemical constituents in the family Rutaceae, to which the commercial citrus varieties belong, see Gray and Waterman (161) and Stanley and Jurd (165). The biogenesis, structural diversity, and distribution of simple furano- and pyranocoumar-

bCH3

Psoralen

Bergapten

bCH3

Xanthotoxin

lsopimpinellin

Figure 9 Major linear furanocounmins found in food plants.

Naturally Occurring Toxic Chemicals in Foods

53

Table 6 Furanocoumarins inLimes

Concentrations (pglg fresh weight) ~~~

~

limes Persian Rind

Key limes

Compound

Psoralen

4/5*

Xanthotoxin Bergapten lsopimpinellin Limettin

7 = ND~, ND one fruit = 0.1 ND 0.1 i 0.1 5.9 f 5.1 20.9 i 34.1a 1.1 f 0.9 128.7 f 32.9 22.0 f 31.4 2.9 f 2.5 53.7 A 14.1 1.7 f 1.3 291.1 f 85.4 310.1 f 136.3

3.9

ND

ND 0.4 f 0.6 1.7 f 2.0 2.8 + 2.1

"can ? standard deviation, N = 8: N = 13. bND = Not detected. Sorrrce: From Ref. 181.

ins in the family Rutaceae are described by Gray and Waterman (161). Herniarin and 7ethoxycoumarin were the most active coumarins against yeasts, molds, and bacteria (165).

B. Limes Sams reported 11 cases of dermatitis from lime oil, of which three were documented cases of dermatitis caused by limeade preparation (174). Limes contain the photosensitizing compounds psoralen, bergapten, and xanthotoxin (Fig. 9). Limes also contain isopimpinellin and limettin (161). However, isopimpinellin is not a photosensitizer (175, 176). Limettin is 1/200 as photoactive as bergapten on rabbit skin (177). Contact dermatitis and photodermatitis have been described in children handling limes (178, 179) (Table 6) and in children making limeade from limes (180, 181). The culprit compound appeared to be bergapten (181), but limettin was present in lime rind at about 300 ppm. Interaction assays between photoactive compounds should be conducted.

IV. CRUCIFERS (CRUC/F€RA€,BRASSICA) Cruciferous vegetables (Cruciferae, Brassica) contain natural compounds that exhibit a variety of biological activities. In ancient times, these crops were cultivated primarily for medicinal purposes (182). The first adverse biological activity investigated was their goitrogenic activity (6, 183). Plants that contain natural goitrogens and belong to this group of vegetables are listed in Table 7.

A.

Goitrogens

As early as 1928, laboratory animals fed cabbage were induced to develop goiters (184, 185). Also, lambs from ewes that were fed plants containing goitrogenic components died. Experiments with rats and guinea pigs in 1964 showed that cabbage has a marked goitrogenic capacity (186). The first goitrogen isolated from cabbage was thiocyanate, but the concentration found in cabbage could not explain the total observed effects (187).

Beier and Nigg

54

Table 7 Plants Containing Goitrogenic Compounds nameCommonname

Latin Beta vulgaris var. ciela Brassica canlorapa Brassica hirta Brassica napus Brassica nigra Brassica oleracea var. acephala var . bottytis var. capitata var. gemmifera var. napobrassica Brasslca pekinensis Brassica rapa Glycine max Linum usitatissimum Juglans regia' Arachis hypogaea"

Chard Kohlrabi White mustard seed Rapeseed or meal Black mustard seed Kate Broccoli Cabbage Brussels sprouts Rutabaga Chinese cabbage Turnip root Soybean Flax Walnut Peanut

'See Ref. 183. Source: From Ref. 6.

The goitrogenicity of cabbage and other cruciferous plants can be explained by the combined action of thiocyanate, goitrin, and allyl isothiocyanate (188). These compounds are hydrolyzed enzymatically from various glucosinolates (1 89). Therange of glucosinolate concentrations found in various cruciferous vegetables is listed in Table 8 (182, 190193). Brussels sprouts have the highest observed levels of glucosinolates, 1430 to 1760 pg/g of fresh sprouts. The types and quantities of glucosinolates have been determined in 22 different varieties and various head sizes of cabbage (193). The total glucosinolate concentration was 663 pg/g of fresh cabbage. The highest total glucosinolate concentraTable 8 Glucosinolate Content of Cruciferous Vegetables Concentration (pg/g) no.

ReferenceRange

Mean Vegetable Broccoli 1590 sprouts Brussels Cauliflower Red cabbage

740

2000 480 770

320

White cabbage (kraut) 61

0 890

White cabbage (market)

530 650

450-1 480 1430-1 760 600-3900 270-830 470-1 240 160-460 430-760 670-1 020 260- 060 1 300-1 070

190 190 191 190 192 190 192 193 192 193

Naturally Occurring Toxic Chemicals in Foods

55

tions found in four cabbage varieties, Red Hollander, Savoy Perfected Drumhead, Wisconsin Hollander,and Stonehead, were 1203, 1288, 1014, and 1065 pg/g of fresh cabbage, respectively (193). Goitrogenic compounds also are found in rapeseed. Rapeseed meals are used in animal feed because of the lower glucosinolate content in newer varieties (194). The enzymatic degradation product of progoitrin, 5-vinyl- 1,3-oxazolidine-2-thione (5-VOT), is goitrogenic, produces antinutritional effects, and inhibits synthesis of thyroid hormones, which causes metabolic disturbances (182). Therefore, its use in animals or indirect consumption by humans presents a potential danger (194).

B. CarcinogenicityModulation 1. Protection from Cancer Rabbits fed cabbage leaves in 1931 survived a lethal dose of uranium (195). This led to the epidemiological conclusion in the 1970s and 1980s that cruciferous vegetables can provide protection from cancer. Indoles in vegetables of the Brassica genus were known to inhibit carcinogenesis in experimental animals. Three 3-substituted indoles [indole-3carbinol (I3C), 3-indolylacetonitrile, and 3,3’-diindolylmethane] (Fig. 10) are inhibitors of induced cancer (196). These three indoles are produced by enzymatic hydrolysis of indolylmethylglucosinolate (glucobrassicin) by the pH-dependent plant enzyme myrosinase following disruption of plant material (197). Nearly 80 naturally occurring glucosinolates have been described. Isothiocyanates or nitriles formed from the glucosinolates are dependent on the type of plant material and the treatment of the material prior to and during hydrolysis (1 98). Seven isothiocyanates were tested for mutagenicity on S. pphimwiuwz TAlOO and all tested positive, with allyl isothiocyanate having the highest potency. Allyl isothiocyanate glucoside (sinigrin) showed an equivalent mutagenicity potency to allyl isothiocyanate itself. Thiocyanates were found to be nonmutagenic, while isocyanates showed mutagenicity on S. ~yphimzlr - i u n z TAlOO strain even without activation (199). Sedation, ataxia, loss of righting reflex, and sleep were induced in rats by 3-indolylacetonitrile and I3C. Phenylpropyl isothiocyanate and allyl isothiocyanate were not teratogenic to rat fetuses, but they did cause embryonal death and decreased fetal weight (200). Animals fed diets high in cruciferous vegetables and then exposed to various carcinogens expressed lower tumor yields and increased survival rates (201-203 j. Arylalkyl isothiocyanates have been determined to be inhibitors of lung tumorigenesis induced by the tobacco-specific nitrosamine 4-(methylnitrosamino)-l-(3-pyridyl)-1-butanone (NNK) in rats and mice (204-206). It now is known that manydrugs and other chemicals induce metabolizing enzymes. Animal feeding studies have demonstrated induction of mixed-function oxidases (MFOs) in rats fed Brussels sprouts or cabbage (207) and cauliflower (208); the indoles present in these vegetables were shown to cause induction of metabolizing enzytnes (197, 209). Especially noted is induction of the intestinal aryl hydrocarbon hydroxylase (Ah) system (207, 210). The Ah receptor is involved in the induction of cytochrome P-450 -I A1 and A2, and other enzymes that participate in xenobiotic metabolism (311). 13C isolated from Brassica olerzrcea var. gemmifercl cv. Jade Cross was a significant inducer of hepatic and intestinal MFOs (197). 13C was shown to enhance the activities of rat intestinal Ah and ethoxycoumarin 0-deethylase, which are capable of metabolizing benzo[n]pyrene and other xenobiotics. In addition, both hepatic and intestinal glutathione S-transferase and

Beier and Nigg

56 NOS03K

QLT?T

glucose

I

H

lndolylmethylglucosinolate

>?-

I

H

3-lndolylacetonitrile

.+

sulfate, & sulfur

glucose,

& sulfate

lndolylrnethyl isothiocyanate

Indole-3-carbinol 3,3’-Diindolylmethane

Figure 10 Enzymatic hydrolysis of indolylmethylglucosinolate. (Adapted from Ref. 197.)

microsomal epoxide hydrolase of the small intestine were induced by I3C. Hepatic cytochrome P-450 was increased by Brussels sprouts and 13C (212). A diet containing Brussels sprouts and cabbage increases the apparent metabolic clearance rate of antipyrine, phenacetin (213), and acetaminophen, while also enhancing its glucuronide conjugation in humans (214). I3C administered orally to humans increased estradiol 2-hydroxylation (215). The effect of 13C on estradiol 2-hydroxylation is similar to that caused by smoking. Epidemiological studies indicate that colon cancer risk is higher in individuals who ate fewer cruciferous vegetables (216) and consumed more fat (2 17-222). Epidemiological studies of people who had gastric cancer also suggested that those who ate cruciferous vegetables were protected (223-225). Other epidemiological studies indicated a lower incidence of breast cancer (226,227) and prostate cancer (227,228) in vegetable-consuming populations. Evidence from an epidemiological case/control study of diet and cancer

Naturally Occurring Toxic Chemicals in Foods

57

also suggested that consumption of cruciferous vegetables was associated with a decreased incidence of cancer (229). Recently, approximately 200 studies that examined the relationship between fruit and vegetable intake and cancer of the lung, colon, breast, cervix, esophagus, oral cavity, stomach, bladder, pancreas, and ovary were reviewed (230). A protective effect of fruit and vegetable consumption was found in 82% of the studies. For most types of cancer, persons with low fruit and vegetable intake experienced approximately twice the risk of cancer compared with those people with high intake (230). The public has been advised by various studies to include more cruciferous vegetables, such as cabbage, broccoli, Brussels sprouts, kohlrabi, and cauliflower, in their diets (230-234). Today, some physicians are making the same recommendation. 2. Promotion of Cancer Unfortunately, the early feeding experiments generally used only one type of protocol; cruciferous vegetables or 13C was given prior to or during administration of a carcinogen. When this protocol was changed so that a carcinogen was given before consumption of cruciferous vegetables or I3C, higher cancer rates were obtained in laboratory animals treated with either cruciferous vegetables or I3C. When 13C was given to trout before aflatoxin B I, the trout were protected against liver carcinogenesis (235). However, when aflatoxin B I was given before I3C, hepatocarcinogenesis was strongly promoted (236). This promoter effect also was observed with 1,2-dimethylhydrazine enhancement of colon cancer in rats (237). 13C was mostresponsible for tumor morbidity and appears to promote tumorigenesis by inducing Ah receptor activity. Colon tumor incidence increased when 1,2-dimethylhydrazine was administered to mice fed a diet containing cabbage (238). The level of cabbage used in these studies was comparable with human intake. Feeding cabbage to hamsters elevated the incidence of gallbladder adenocarcinoma, plus feeding high-fat diets elevated pancreatic ductular carcinoma in hamsters administered N-nitroso-bis-(2-oxopropyl)amine (BOP). Skin papilloma, initiated by 7,12-dimethylbenz[n]anthracene(DMBA) and promoted by 12-0-tetra-decanoylphorbol-13-acetate (TPA), increased when mice were fed diets containing 10% dried cabbage (239). Possible biochemical mechanisms behind the promotional effects of 13C are under study. I3C, given intraperitoneally, does not induce hepatic ethoxyresorufin 0-deethylase (EROD) activity. Given orally, it does induce EROD (196). Acid treatment of I3C, under conditions similar to stomach acid conditions, produced a reaction mixture that induced EROD by either the intraperitoneal or oral route. Chromatographic separation of the acid reaction mixture suggested that at least four 13C condensation products will induce EROD (240). The mechanism by which 13C and its analogs induce cytochrome P-448-dependent monooxygenases is mediated via condensation products generated in the acidic conditions of the stomach (196). Glucobrassicin (241) and neoglucobrassicin (242) are proposed as the sources of 3indolylacetonitrile, I3C, and other simple indolic compounds. The glucobrassicin content of fresh Brussels sprouts varies from 220 to 11 10pg/g (191). The pH-dependent enzymatic hydrolysis of these indole glucosinolates by myrosinase produces 13C and a series of simple indoles (197, 243) (Fig. 10). I3C, 3-indolylacetonitrile, and the well-known MFO inducer, 3,3’-diindolylmethane, from cabbage and cauliflower were all demonstrated to induce the Ah receptor (197). Since compounds such as indolo[3.2-b]carbazole (ICZ) (Fig. 11) can be generated from 3,3’-diindolylmethane in the presence of acid, air, and

Beier and Nigg

58

LT

CT

H

ICZ Figure 11 Condensation products of indole-3-carbinol in acidic conditions similarto those in the stomach (LT = 2-[indole-3-ylmethyl]-3,3’-diindolylmethane: CT = 5,6,11,12.17,18-hexadrocyclonona[ 1.2-b:4.5-t7’:7.8-b’]triindole;ICZ = indolo[3.2-b]carbazole).

light (244), it was postulated that ICZ may be present (196), ICZ was also the most active of all the indoles studied for inducing the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) receptor (245). The three most prevalent UV-absorbing compounds in the reaction mixture of acidtreated 13C are 2-(indol-3-ylmethyl)-3,3’-diindolylmethane(LT), 5,6,11,12,17,18-hexahydrocyclonona[ 1.2-b:4.5-b’:7.8-b’’]triindole (CT) (Fig. 1l), and 3,3’-diindolylmethane (Fig. 10) (246). Molar yields of the three were in the range of 2-6% of the original amount of I3C. The presence of LT and 3,3’-diindolylmethane was consistent with previous findings (247). Upon further analysis, ICZ was shown to bepresent in theacid condensation reaction mixture (248). ICZ is produced from 13C in yields on the order of 0.01% in vitro and, after oral intubation, in vivo. The binding affinity of IC2 is a factor of 3.7 X 10” lower than that ofTCDD. The most potent Ah receptor agonist identified in the acid condensation reaction mixture is ICZ. ICZ and related condensation products appear responsible for the inducing effects of dietary I3C. Due to the higher yields of the weaker-binding oligomers, ICZ appears of roughly equal importance to other oligomers in the inducing activity of the mixture. TCDD has well-known and established activities as both an anti-initiator and as a promotor of carcinogenesis. Similar effects are observed for the cancer-modulating activity of I3C. 13C or TCDD given before a carcinogen protects against carcinogenesis; when either is given after a carcinogen, it strongly promotes carcinogenesis.

Naturally Occurring Toxic Chemicals in Foods

59

The Ah receptor bindings of ICZ and TCDD are similar in all respects. A 100-g portion of Brussels sprouts could provide a dose of 0.256-1.28 pg ICZ. This dose is considerably in excess of the maximum acceptable daily human dose for TCDD, which is 400 fg/70-kg person, established by the U.S. Environmental Protection Agency (EPA). However, there may be a number of factors that could lower the relative hazard or benefit as well as the half-life of ICZ compared to TCDD. Bjeldanes et al. concluded that it appears unlikely that normal levels of ICZ in the diet are a significant hazard compared with the benefit of the micronutrients in Brassica vegetables (248). 3. Induction of Protective Enzymes Zhang et al. isolated a single isothiocyanate, sulforaphane (Fig. 12),from SAGA broccoli that was the major inducer of phase I1 protective enzymes (249). The assay used resulted in the determination of quinone reductase in Hepa IC lc7 murine hepatoma cells. However, Zhang et al. did not include references that describe 13C as themajor component of cruciferous vegetables responsible for the observed biological activities of cruciferous vegetables in animals (see Sec. 1V.B.l and IV.B.2) (249). Since similar effects are observed for both cruciferous vegetables and I3C, an interesting question is, What role does sulforaphane actually play in protection against carcinogens with respect to 13C in cruciferous vegetables? Cruciferous vegetables, I3C, or TCDD given before a carcinogen protects against carcinogenesis, but when any one of the three is given after a carcinogen, each promotes carcinogenesis.

V.

FRUITS AND VEGETABLES(FLAVONOIDS)

The amount of flavonoid literature is enormous. We refer the reader to Refs. 250-255 for a general view of flavonoid chemistry, biochemistry, and distribution. A mass spectral analysis study of 79 flavonoids provided data on 26 compounds that were not found in the National Institute of Standards and Technology database (256). McClure reviewed the function and physiology of flavonoids (257). Plant flavonoid content may be influenced by light, water, temperature, sugars, mineral nutrition, mechanical damage, pathogens, plant growth regulators, and various other chemicals (257-260). Microorganism-resistant cultivars may contain higher flavonoids than susceptible cultivars (261), and flavonoids may vary during a plant’s life cycle (262). Flavonoids may be localized in plant tissues and cells and secreted in various exudates. They may function as antioxidants, enzyme inhibitors, pigments for light absorbance, visual attractants for pollinators, light screens, promoters or inhibitors of plant growth, plant growth regulators, legume Rhizobimz root nodule gene inducers (263, 264), phytoalexins, analgesic agents (265), and plant morphogenic and sex-determination agents (257).

and

60

Beier

Nigg

Kiihnau (266), Hackett (267), and Adzet and Camarasa (268) reviewed flavonoid metabolism. Less-hydroxylated or -methoxylated flavonoids are more active biologically and are more resistant to intestinal microflora metabolism. An exception is the biflavan, 3,3’,4’,5,7-pentahydroxyflavonoid (cyanidin) (see the flavonoid general structure in Fig. 13), which is resistant to gut microflora. The flavonoids are metabolized to phenolic acids or lactones in the gut, are absorbed by the gut as aglycones, may be excreted unchanged in bile, and may be metabolized by the liver. They may be oxidized, reduced, methylated, and conjugated. Although most of these experiments have been in small animals, human experiments suggest similar processes (267). Overall, the absorption, metabolism, and excretion of flavonoids in any animal, particularly humans, have been poorly studied. No study has accounted for the entire dose. In radiolabeled studies, the form of the radiolabel recovered, in most cases, was not determined. Radiochemical purity of starting materials was not determined. Elimination halflives are helpful but are not necessarily related to dose. There are no mass-balance studies.

6

4

Flavonoid general structure

Q

OH

0

Flavonols

0

Flavones

AH

Q

Leucoanthocyanins

Flavanones

fJ

OH

Q 0O

H

Flavanonols

0

0 OH

Catechins

Anthocyanins

Figure 13 Flavonoid general structures.

Naturally Occurring Toxic Chemicals Foods in

61

The pharmacokinetics of absorption, metabolism, and excretion of flavonoids is critical for estimating benefits and risks.

A.

Dietary Flavonoids

Flavonoids usually are conjugated to a sugar and are widespread in the plant kingdom (253, 269). The occurrence of the flavonoid aglycone is less pronounced compared with the carbohydrate moiety of the conjugates and appears to be associated with secretory structures and lipophilic plant products (see Ref. 270 for a review). Pierpoint lists several difficulties in estimating flavonoid intake (271-273). Many compounds are involved. Flavonoid content varies by season, plant cultivar, plant maturity, and plant condition. National diets vary, so an estimate for the United States may not apply to other countries and cultures. Kuhnau published an estimate of the total U.S. flavonoid daily intake per person of 1020 mg/day (winter) and 1070 mg/day (summer) (266). Of these totals, 41% (winter) and 39% (summer) are from cocoa, cola, coffee, beer, and wine. Fruit juices contribute an additional 26% (winter) and (29% (summer), followed by spices at 16% (winter) and 15% (summer). These three food groups contribute 83% of the total daily U.S. intake per person of flavonoids in winter and summer. Pierpoint discussed culture differences for flavonoid intake (272, 273). In the United Kingdom, for instance, the average tea consumption of 4.7 cups daily per person would provide about 900 mg flavonoids, whereas the total estimated U.S. daily consumption per person from all sources is about 1000 mg (266). Dependent on diet, total flavonoid content in some cultures may be 2000-3000 mg/day (273). Many vegetables contain flavonoid compounds: bell pepper, broad bean, broccoli, cabbage (regular and red), chive, endive, garlic, green potato, horseradish, kale, kohlrabi, leek, lettuce, onion, and radish (274-277). Most of these vegetables contained less than 100 pg/g of either quercetin and/or kaernpferol (Fig. 14). However, the colored, outer skin of onion contained up to 65,000 pg/g of quercetin (276), broad bean pods 1340 pg/g of quercetin (276), and green endive 9400 pg/g of quercetin (275). Shallots contained more than 800 pg/g of total flavonoids; 20 cultivars of yellow and red onions contained 60-1000 pg/g, but flavonoids were not detected in white onions (278). Quercetin and kaempferol have been quantified in the following fruit: apples, apricot, bilberry (wild and cultivated), blackberry, black current, highbush blueberry, cherry, cranberry, currant (red and white), elderberry, gooseberry, grape juice, pear, peach, plum, prune (dry plum), apple juice, and quince (276, 279-281). These mostly contained less than 75 pg/g except for apples (about 100 pg/g) (276), bilberry (about 125 pg/g) (276), cranberry (about 175 pg/g) (279), elderberry (about 150 pg/g) (276),prunes (22,000 pg/ g) (282), and quince (about 390 pg/g) (276). Other flavones found in fruit include catechin, in the apple, apricot, cherry, peach, and plum (283);naringin, in various citrus including juice (284); and 5,7-dihydroxychromone, eriodictyol, and luteolin, in peanut hulls (285). Flavonoid content of varieties of the same fruit differ as well as flavonoid content of plant' parts at different stages of maturity. Light conditions affect flavonoid content (276). For example, covering cauliflower curds reduced floret flavonoids 20-40% and improved the quality of the cauliflower (286). Plant disease status affects flavonoid content (287, 288). Processing, shipping, and handling also may affect final flavonoid levels. Some human flavonoid consumption undoubtedly comes from food contamination. Commercial grain may be contaminated with toxic weed seeds, sicklepod (Cassia obtusifolicr), jimson weed (Datum stranzoniunl), velvetleaf (Abzltilo~theophrasti), and morning

Beier and Nigg

62

Kaempferol

QH

PH

OH OH

OH

Myricetin Figure 14 Mutagenicflavonols from fruits andvegetables.

glory (Ipomoea species) (289). Sicklepod seeds contain anthraquinone derivatives, P-sitosterol, and flavonoids, UV-quenching compounds, and fluorescent blue compounds of unknown structure (289). Jimson weed contains tropane alkaloids; velvet bean contains delphinidin, quercetin, catechin, myricetin (Fig. 14), (- )-epicatechin, and cyanidin (291). Morning glory seeds contain chlorogenic acid (Fig. 3) (289). Range plants also may contain flavonoids. Gutierre,-in microceyhnln A. Gray (broomweed, perennial snakeweed, broom snakeweed, stinkweed, turpentine weed) consumption by cattle in the American Southwest is associated with abortion, placental retention, hemorrhage, and nonsurviving weak offspring (292-294). Over 20 oxygenated flavonol methyl ethers were isolated from the Gutierrezicr species (294). Soybeans contain daidzein (aglycone of daidzein), genistein (aglycone of genistin) (Fig. 15) (295), and 3-@malonyl and 3-acetyl-isoflavones (296). These compounds are bitter or astringent (296). Daidzein and genistein increase due to P-glucosidases in soybean during the processing of soy milk. On a dry soybean basis, these compounds increased

Naturally Occurring Toxic Chemicals in Foods

63

OH

Diethylstilbestrol

Equol

HO

OH

OH

Daidtein

Genistein

OH

Formononetin

Coumestrol

Figure 15 Structural formulas of equol, diethylstilbestrol, diadzein. genistein, formononetin, and coumestrol.

from about 50 pg/g to 350 pg/g at pH 6.0 (close to the pH of soy milk) and 20°C in the soak step of processing (295). In the soybean plant, daidzein, genistein, and coumestrol (Fig. 15) increased in the roots over a 12-day period after transplanting and inoculation with Br.ndyrlzizobiunzjclporzicum. Nitrogen application decreased the isoflavone (Fig. 16) concentration in roots (297, 298). Conjugates of daidzein and genistein are selectively

Figure 16 Isoflavonoidgeneralstructure.

64

Beier and Nigg

excreted into root and seed exudates at a level of 1-10 yM. Their levels in the seed may be more than 1000 pg/g seed tissue and it is postulated they may act as signal molecules in chemoattraction of nodule-forming bacteria (299). Subterranean clover (Trifolium subterrcrneum L.) is a forage legume widely grown in Mediterranean climates (300). Identified isoflavones in subterranean clover include biochanin A, formononetin, genistein, and daidzein (Fig. 15). Of four cultivars, only one showed an isoflavone content difference based on harvest date. Total isoflavones were almost unchanged from the comparison of fresh to frozen samples, but dried samples showed a 30-50% decrease in isoflavone content (300). Production of isoflavones was induced in the bean (Phaseolus vzrlgnris L.) by ozone, SO:, and several herbicides (301). 6. BiologicalEffects of Flavonoids There are perhaps more good health claims for flavonoids than there are adverse effects reported because humans perceive the healthy aspects of citrus, pome fruits, and vegetables. The U.S. surgeon general and the National Academy of Sciences have strongly recommended consuming more fruits and vegetables. The Committee on Diet, Nutrition, and Cancer, National Research Council, emphasized the importance of including fruits (especially citrus fruits), vegetables (especially carotene-rich and cruciferous vegetables), and whole-grain cereal products in the daily diet (232, p. 15). Kuhnau (266), Singleton (302), and Attaway (303) reviewed the nutritional effects of flavonoids. Flavonoids are considered “semiessential’ ‘ food components and not harmful. Flavonoids are antioxidants (304). Quercetin, myricetin (Fig. 14), quercetagetin, and gossypetin are the best antioxidants; catechin has some activity. Hesperidin is inactive. Daidzin, genistin, malonyl-daidzin, and malonyl-genistin were antioxidants (262, 305). Morin, quercetin, myricetin, and fisetin were the most active inhibitors of induced lipid peroxidation in the presence of ferrous ions (306). Diosmetin, apigenin, hesperetin, naringenin, and 4’,5,7-trihydroxyflavone(all lack the 3-OH group) (Fig. 13) were least active. With ascorbic acid as the inducer, 3-hydroxy-flavone, fisetin, taxifolin, (+)-catechin (Fig. 13), quercetin, myricetin, and morin were the most effective inhibitors of lipid peroxidation (306). These all have a 3-OH group. Isovitexin, a glycosyl flavonoid from rice hull, is a better antioxidant than BHA (butylated hydroxyanisole) and a-tocopherol (307). Isovitexin is absent in rice seeds that deteriorate or rot. Only long-lived rice seed hulls contain isovitexin. Epicatechin 3-0-gallate and other procyanidins from grape seeds were found to be potent, oxygen-free, radical scavengers. The best scavenger was procyanidin B23’0-gallate (308). Some flavonoids can chelate metal ions, particularly copper (266, 309). Copper flavonoid complexes inhibit hyaluronidases, stabilize structural proteins, and strengthen fragile membranes (266). Flavonoids hydroxylated in the 5, 3’,4’ or 5, 3’,4’,5’ positions (Fig. 13), may be competitive antagonists to catecholamines like epinephrine and norepinephrine and extend the effect of these compounds. Flavonoids also extend the action of vitamin C and, at one time, were referred to as vitamin P (310). The term vitamin P was replaced by bioflavonoid. Vitamin P was a mixture of eriodictyol and hesperidin. The interaction between vitamin C and flavonoids is considered to be an antioxidant effect (for reviews, see Refs. 31 1 and 312). Flavonoids reportedly affect glucose uptake by lymphocytes and fibroblasts, and for their immune functions act as smooth muscle antispasmodics, and anti-inflammatory agents. They inhibit platelet aggregation, act as antiviral agents, are synergistic with other

Naturally Occurring Toxic Chemicals in Foods

65

antiviral agents, and are bacteriostatic. Flavonoids also are potent multifunction oxidase inducers (thus increasing metabolism of xenobiotics), correct abnormal capillary permeability and fragility after X-radiation and a variety of diseases, and act as anticancer and antimutagenic agents. Quercetin (Fig. 14) is the principal flavonoid tested for these effects (266, 303, 309, 310, 312). Quercetin at 25,000 pg/g in laboratory chow or 70 pg/g in water prevented diabetic cataracts in the degu (Octodon degus), a rodent native to the Andes of South America, possibly through inhibition of lens aldose reductase (313, 314). Flavonoids inhibit a variety of enzymes in vitro that are required for normal physiological function (3 12, 3 15-3 17). Some of these enzymes are involved in axonal transport, basophil/mast cell secretion, cell locomotion/chemotaxus, DNA synthesis, endo-/exocytosis, insulin secretion, intestinal chloride ion secretion, membrane phosphorylation, microtubular dissociation, mitogenesis, neurotransmitter release, platelet function, and smooth muscle contraction (312, 317). Flavonoids may interfere with thyroid function (T,-T, balance) (3 18). A particularly fascinating biological activity of quercetin is its ability to inhibit heatshock protein by interacting with heat-shock factor. This activity is of practical consequence in reducing the heat tolerance of tumors during hyperthermic therapy (3 19). Although many foods have notbeen shown to be mutagenic in microorganism assays, red wine, grape juice, instant coffee, strawberries, raspberries, peaches, raw onions, raisins, and grapes displayed potent mutagenic activity and the primary natural mutagen appeared to be quercetin (Fig. 14) (320-323). In 1977, Bjeldanes and Chang reported that quercetin, a large fraction of the total flavonoids in the daily diet (324), was mutagenic without cytochrome P-450 activation, but was more mutagenic with activation (325). In further studies, quercetin and myricetin (Fig. 14) were mutagenic without activation; kaempferol (Fig. 14) was mutagenic only with activation (326, 327). Quercetin was mutagenic to mouse cell lines (328). Flavonoid content correlated with the mutagenicity of nutritional supplements and tobacco (snuff) containing rutin (326). Of the 70 naturally occurring and synthetic flavonoids tested, 33 were positive in the Ames assay (329). All active flavonoids have maximum active response toward S. ~ p h i m u r i u nstrain ~ TA98 and a highly significant response toward strain TAlOO (309, 329). The flavonoid structural features essential for mutagenic activity in S. typhin~uri~m strains TAlOO and TA98 were determined, and only the flavonols (3-hydroxyflavones) (Fig. 13) appeared to be mutagenic (327). Structural requirements for mutagenic activity appear to be: (1) a free 3-hydroxyl group; (2) a 2,3 double bond; and (3) a 4-keto group (Figs. 13, 14). However, there are flavonoids without the 3-OH group that are mutagenic toward the TAlOO strain, but not strain TA98 (309). Quercetin is the most-active flavonoid mutagen with strain TA98. Oxygen, tyrosinase, and alkaline pH irreversibly inactivate the mutagenicity of quercetin with strain TA98 (330). Quercetin also was positive in the SOS chromotest, apparently without activation (33 1). Norwogonin and sexangularetin are more mutagenic than quercetin with strain TA100. These flavonoid mutagens require activation with the S9 or cytosol cell fraction, and have hydroxy- or methoxy-substitution at positions 5,7, and 8 of the A ring (see Fig. 13). The B ring and the 2, 3 positions apparently are not involved in this second class of flavonoid mutagens (309j. Flavonoids may be responsible for the mutagenicity of weed seeds that contaminate grain (289). The mutagenicity of quercetin probably prompted its inclusion in the National

Beier and Nigg

66

Toxicology Program’s testing regimen. Quercetin was mutagenic in Snlrnonellcr assays and cytogenic in Chinese hamster ovary cells (332). Quercetin subsequently was shown to exhibit some carcinogenicity in male mice because of increased renal tubular cell adenomas when fed at 40,000 ppm (333, 334). Cancer studies on flavonoids have been reviewed (309, 335, 336). Quercetin (Fig. 14) was positive for intestinal tumors, bile-duct tumors, bladder tumors, hepatomas, and liver preneoplastic foci in three rat dietary studies. In 10 other rat/mouse/hamster dietary studies, there was no increase in carcinomas (309, 336). Rutin, kaempferol (Fig. 14), tiliroside, and catechin (Fig. 13) were negative (309, 329). In chromosomal aberration tests, quercetin has been both positive and negative (309, 329, 337). Flavan-3-ols, (+)catechin, (-)-epicatechin, (+)-gallocatechin, (-)-epigallocatechin-3-O-gallate, and procyanidin B-1 and C-1 break double-stranded DNA in the presence of cupric ions (338). Quercetin at 0.1% in the diet reduced the life span of “shorter-living” male mice (339). Flavone acetic acid, a synthetic compound, is used as an anticancer drug (340, 341), though not as a single agent (342). A wide variety of flavones have been synthesized to obtain cytotoxic agents (343). Various flavonoids are antimutagenic agents (344-346). Quercetin reduced mortality and the cytotoxic effects of the T-2 mycotoxin in mice (347). Various flavonoids inhibited the mutagenicity of aflatoxin B 1 toward S. ?yphimurilrm strains TAlOO and TA98 (348). Some flavonoids have been found to be antitumorigenic, antimutagenic, and anticarcinogenic (266, 278, 316, 349-353). Flavonoids appear to provide a cancer protective effect at the cellular and molecular level (278, 351), and also by reducing the bioavailability of carcinogenic compounds (351, 353). It is important to note that mutagenicity testing is usually with a single compound whereas “anti” effects usually involve use of a known carcinogen plus a flavonoid. Canada et al. challenged isolated guinea pig enterocytes with kaempferol, quercetin, and myricetin (Fig. 14) (354). All three compounds produced cellular damage at 450 pM. Quercetin and myricetin appeared to be more toxic than kaempferol. These authors suggested that flavonoids might exacerbate or cause inflammatory bowel diseases (354). Kumar et al. administered kaempferol orally at 250 mg/kg body weight/day for 60 days to male rats (355). Spermatids were reduced by 73.7%. Mature and immature Leydig cells decreased by 39.2% and 46.6%, respectively. Testicular cholesterol increased and androgen-dependent sialic acid and protein declined in the testes, epididymides, and sex accessory glands (355). Shore and Lyttle studied the inhibition of rat uterine peroxidase, an enzyme that increases in the uterus in response to estrogen (356). Diethylstilbesterol, genistein (isoflavone) (Fig. 15), and zeralenone and zearalenol (Fig. 17) were competitive inhibitors; coumesterol (isoflavone) (Fig. 15) was a noncompetitive inhibitor of rat uterine peroxi-

Zearalenol

Zearalenone Figure 17 Natural estrogens that inhibit rat uterine peroxidase.

Naturally Occurring Toxic Chemicals in Foods

67

dase. Coumesterol was 2-6 times as inhibitory to peroxidase as diethylstilbestrol (Fig. 15), whereas genistein was 25 times less active than diethylstilbestrol (356). Flavonoids have been linked with abortion in cattle in theAmerican Southwest from consumption of the genus Gutierrezin (294). The isoflavones (Fig. 16) of subterranean clover, formononetin, biochanin A, genistein, and daidzein (Fig. 15) are associated with an infertility syndrome in sheep (357-360). Estrous ewes fed oats or subterranean clover averaged 17,160 and 350 sperm, respectively, per fallopian tube 24 hr after mating. The percentages of motile sperm recovered from the cervix and ova with sperm attached to the zona pellucida were lower in ewes fed clover (361). The isoflavone infertility problem has prompted immunization attempts against genistein and equol (Fig. 15) with temporary success (362). Equol has been identified in the urine of pregnant mares (363), goats (364), cows (365), hens (366, 367), sheep (368, 369), and men, women, and rats (370). The metabolism of formononetin (Fig. 15) and biochanin A in bovine rumen fluid has been studied (371). Analytical methods for isoflavones in soy protein (372) and in human urine (373) are available. Equol (Fig. 15) and other phytoestrogens possess weak estrogenic activity (374377). Estrogenic responses to isoflavones vary by species and, for bioassays using mice, by strain (378). Human volunteers excreted large quantities of equol, a phytoestrogen, after consuming 40 g of textured soya for 5 days. Fecal flora also were incubated with textured soya and intestinal microbes produced equol. Equol excretion exceeded 6 mg/ day in one subject after a 40-g soya meal. Estrone-glucuronide, the principal urinary estrogen in the follicular phase of women, is excreted at 2-27 pg/day (379). Urinary excretion of equol in humans not consuming soy products is about 80 pg/day (379). This study is important for several reasons. It showed conversion in humans to a biologically active flavonoid from a dietary source. Equol crossed into the bloodstream in quantity and was excreted as a glucuronide. Phytoestrogens apparently cotnpete for the same estrone binding sites on a-fetoprotein in rats and in humans (380). Phytoestrogens have a wide range of biological activities, from anticancer to antiestrogen effects (380). Flavones and isoflavones (Fig. 16), including genistein (Fig. 15), biochanin A, prunetin, kaemferol, and quercetin (Fig. 14), inhibit tyrosine protein kinase, which is necessary for retrovirus carcinogenicity (381). Genistein induces mouse erythroleukemia cells synergistically with mitomycin C (382). Immunological data on genistein suggest it is a powerful immunosuppressant (383). Flavonoids also affect the immune system and inflammatory cell functions. These effects are reviewed in Ref. 384. Flavonoids also reduced benzo[a]pyrene DNA adduct formation in vitro and also in vivo after animals were fed flavonoids for 2 weeks (385). Perhaps other flavonoids in the diet are converted to biologically active molecules that have yet to be isolated. Increased dietary fat has been linked to prostate cancer and increased plasma levels of male hormones (386). Low-fat diets can influence plasma levels and excretion of estrogens and influence the incidence of breast cancer (387-390). We suggest that a better understanding of the changes in hormone levels caused by bioactive flavonoids and flavonoid metabolites in the vegetarian diet would be a good starting point in searching for ‘‘anticancer nutrients.’’

VI.

HERBS

The use of herbs and herbal preparations has been reviewed with a historical perspective (391). Many people believe that plant remedies are naturally superior to synthetic drugs

Beier and Nigg

68

and that when herbal preparations are used they cannot be harmful to human beings. In 1977, the Consumer Response Corporation reported that a survey showed that the most convincing sales claim to put on a food or beverage label is that it is “natural”; 42% of the consumers surveyed believed that natural products have no adverse effects and are more healthful and safer (392). In thepast two decades, natural foods and herbal medicines have gained substantial popularity in the United States (393). In 1978, the sale of herbs and other related commodities in health food stores alone amounted to $1.1 billion (39 l), and in 1990 was estimated to be approximately $2 billion (394). There is no doubt that some plants do contain biologically active compounds that are medicinally useful. More than 20% of the commercially prepared drugs originate from plants, but these plants contain many active ingredients that also can provoke adverse reactions. Many people, including physicians, are not aware of the dangerous side effects and sometimes fatal adverse reactions that may occur when using these plants (395).

A.

AsianMedicinal Herbs

Throughout history, infectious diseases have been treated with herbal medications, and scientists at present continue to evaluate and identify their active principles. Various biologically active plants and active ingredients in medicinal plants from Aztec-derived Mexican folk medicines to Chinese herbal preparations have been reviewed (396). Of the traditional Chinese medicinal herbs, 178 were investigated for an anti-Bacteroides fragilis substance. B. fingilis is found predominantly in fecal material and produces butaric acid. The bacterium is often obtained from soft-tissue infections. Only one of these herbs, rhubarb root (Rheunz oflcinnle), was found to possess anti-B. fragilis activity. The active substance subsequently was isolated and shown to be 1,8-dihydroxyanthraquinone (397). Also evaluated against 10 microbial pathogens were 18 herbs. Of these, 11 preparations were active against at least one pathogen, six were active against at least three pathogens, and two were active against five pathogens (398). Chinese medicinal herbs, first used more than 2000 years ago, are still used today to treat heart problems (399). When Chinese and Western medicines were combined in the treatment of coronary heart disease in China, a decrease in the mortality rate from 20-30% down to 10-15% was observed. Extracts of 11 out of 27 Chinese medicinal herbs were active against the human imnunodeficiency virus (HIV), and Chinese medicinal herbs appear to be a rich source of drugs for the treatment of HIV (400). Immunomodulatory activity was documented in fractions of Astragalus membrmnceus. Fractions from this Chinese medicinal herb fully corrected an in vitro T-cell function deficiency in cancer patients (401). When 10 Korean medicinal herbs were evaluated for mutagenicity by the Ames test, false-negative reactions were obtained. The substances that inhibited the production of positive reactions were removed through solvent fractionation, which improved the reliability of the results (402). Morimoto et al., while examining 104 medicinal herbs, also found that medicinal herbs contained cytotoxic materials that can limit the applicability of the Ames test (403). Of Pakistani medicinal herbs or mixtures used in treating children, 10 were tested with the Ames test. Extracts of Peshwar (mixture of unknown herbs), Saussuren lappa, Swertia chiraitn, and Skimmia lulrredn were mutagenic. The addition of liver microsomal enzymes increased the activities of two extracts. 1. Adulterated Herbal Preparations There is no doubt that herbal medicines contain effective drugs for specific illnesses. Unfortunately, the herbal medications usually are not prescribed by individuals with the

Naturally Occurring Toxic Chemicals in Foods

69

scientific knowledge of their contents, and the herbs may contain many biologically active components. At times, herbal preparations are laced with drugs to improve their effectiveness (395). The manufacturers of some Chinese herbal medicines for the treatment of arthritis and back pain have adulterated these herbal products with aminopyrine and phenylbutazone (probably to promote the commercial potential of their products). Both compounds are well-known causes of agranulocytosis and have caused many fatalities. Aminopyrine was removed from over-the-counter sale in the United States in 1938 (404). In addition to adulteration by humans, herbal drugs also may contain mycotoxins and mycotoxin-producing fungi (405).

2. DiverseBiological Activities Because of the increased popularity of natural foods, health foods, and herbal products, the public needs to be aware of and concerned about the potential dangers associated with extensive use of these herbal products (406). The biological activities found in a single herb are truly diverse. A given herb usually will contain many components having quite varied, and often opposite, biological activities. At least 25 psychoactive substances have been identified in herbal preparations, and a number of intoxications have resulted from their use. Plants used in herbal preparations that contain psychoactive substances include broom. California poppy, catnip, cinnamon, hops, hydrangea, juniper, kola nut, nutmeg, periwinkle, thorn apple, and wild lettuce (407). Natural pesticides and other biologically active materials in health foods and herbal products constitute a pharmacopoeia of uncontrolled substances in our nation's health food stores.

3. PyrrolizidineAlkaloids Consumption of herbal medicines that contain pyrrolizidine alkaloids may contribute to the high incidence of chronic liver diseases, including cancer, in Asia and Africa (408). In one study, three of 50 medicinal plant species from Sri Lanka contained pyrrolizidine alkaloids (409); these were Crotcdnrin ver'rucosnL., Holarrhenn antidysenterica (L.) Br., and Cassia nuriculata L. In another study of 75 medicinal plants from Sri Lanka, only Crotnlaria juncea L. contained pyrrolizidine alkaloids. Of the other plant species not containing pyrrolizidine alkaloids, three produced hepatic lesions in rats, and two produced marked renal lesions (4 10). Pyrrolizidine alkaloids can cause cirrhosis of the liver and occur in at least eight plant families (41 1). They are genotoxic and mutagenic (412). Pneumotoxicities may result from pyrrolizidine alkaloids (413). These compounds also crosslink DNA (414). A single intravenous (IV) 3.5 mg/kg body weight dose of monocrotaline pyrrole produced delayed pulmonary microvascular leak, interstitial inflammation, and pulmonary hypertension after 14 days in rats (415). A single oral dose of 120 mg/kg body weight of monocrotaline produced a variety of biochemical and pathological liver changes in rats (416). The guinea pig appears to be metabolically resistant to pyrrolizidine alkaloids (417) and male rats are more resistant than females (418). Humans exhibit wide variations in their ability to metabolize these compounds (419). Pyrrolizidine alkaloids contain a basic moiety consisting of one nitrogen at the bridgehead of two five-membered rings (Fig. 18a). Cordell has reviewed the chemistry of these highly biologically active alkaloids (41 1). Indian herbal teas caused hepatic venoocclusive disease in people who had consumed them (420). The herbs, identified as Heliotropium lnsiocnrpum Fisch. and Mey., contained pyrrolizidine alkaloids. The total alkaloid content of this herbal mixture was 0.47%, dry weight. The major compounds identified were heliotrine and lasiocarpine (Fig. 18b), and the minor constituents were europine and

Beier and Nigg

Heliotrine

Lasiocarpine

Figure 18 Pyrrolizidine alkaloid chemical structures: (a), basic structural moiety containing one nitrogen at the bridgehead of two five-membered rings; (b), major compounds identified are heliotrine and lasiocarpine.

heleurine (421). The LDsoof heliotrine is 300 mg/kg body weight, and of lasiocarpine is 72 mg/kg body weight in rats (420). Comfrey (SymphytLlm species) is an herb used as a green vegetable, beverage, or remedy. The leaves and roots of a species from Japan (Synzphytunl offkinale)were hepatocarcinogenic in rats; this species contains at least eight pyrrolizidine alkaloids. Large differences in alkaloid concentrations occur between young and old comfrey leaves. The large, mature leaves have the lowest concentrations (422). The alkaloid contents of dried leaves are 0.003-0.2%, and those of dried roots are 0.2-0.4%. The amount of alkaloid consumed in a cup of comfrey root tea is 12-36 mg. A gelatinous residue forms during the process of making the tea; if it is eaten, as much as 26 mg of alkaloids could be consumed. Reliable data on effects of comfrey on humans are scarce, but available data indicate that the use of comfrey root tea could have serious health consequences (423). Comfrey also may be contaminated with other plants, for instance, deadly nightshade (424). It has been recommended, in no uncertain terms, that comfrey be removed from public consumption (425).

B. OnionandGarlic Historically, people have taken onion and garlic juices as a remedy for a long list of ailments. Both onion and garlic juices can prevent the rise of serum cholesterol after a fatty meal (426). Garlic inhibits lipid synthesis; reverses cholesterol-induced atherosclerosis in rabbits; decreases serum cholesterol, triglycerides, low-density lipids (LDLs), and verylow-density lipids (VLDL); increases high-density lipid (HDL) levels (391); and onion and garlic oils have anticancer activity (427). However, in high doses, wild garlic can cause gastroenteritis, diarrhea, rash, and leukocytosis. Long-term ingestion of wild garlic or onion also will block iodine uptake by the thyroid (395).

Naturally Occurring Toxic Chemicals in Foods

71

Epidemiological studies have demonstrated an inverse correlation between the dietary intake of garlic and onion and stomach cancer risk (428, 429). The antineoplastic effect of these plant oils may be due to the presence of an allyl group containing organosulfur compound (430-432). Eight organosulfides were tested for their inhibitory effects on benzo[a]pyrene-induced neoplasia of the forestomach and lung in the mouse (431). Of the organosulfides tested, diallyl sulfide (DAS), allyl methyl trisulphide, allyl methyl disulfide (AMD), and daillyl trisulfide inhibited benzo[cr]pyrene-induced forestomach tumors, and DAS and AMD also inhibited pulmonary adenoma (43 1). Studies were undertaken to elucidate the mechanism of the antineoplastic effect of DAS (427). It was observed that DAS exerts an antineoplastic effect by modulating glutathione (GSH) dependent detoxification enzymes. Stomach GSH peroxidase activity was increased in a dose-dependent manner. The GSH peroxidase activity also was elevated in the lungs of female CD-1 mice treated with DAS, but it was not dose dependent (427).

C. Yarrow Herbs and natural herbal preparations, in most cases, do not have just a single active component; rather, they have an elaborate array of biologically active components. For example, over 40 indigenous naturally occurring chemical components of the herb yarrow (Adzillen rnillefoliurn) and their biological activities are listed in Table 9 (433, 434). Yarrow is reportedly a hemostatic herb, but it also contains coumarins, which are anticoagulants (433). Thus, varied biological activities can be manifested from crude plant preparations when the proportions of indigenous chemicals change as a result of variable growing conditions or crop treatments during and after the growing period. D. HerbalTeas 1. Chamomile Chamomile tea is a herbal drink commonly sold in supermarkets; people may have allergic or anaphylactic reactions to it. Allergens from chamomile flower heads cross-react with ragweed, chrysanthemums, or other species of the family Compositae (406). The MexicanAmerican population in southern Colorado commonly treat childhood illnesses with the tea. However, chamomile is low insodium, and its continued use without other food intake can cause water intoxication with subsequent hyponatremic seizures (435). Excessive use of chamomile also can cause diarrhea (395). 2. Sassafras Root Herbal teas are used by many people for medicinal purposes as well as for enjoyment. The carcinogenicity of safrole, the natural flavor in root beer, was discovered in 19601961. This discovery led to its banning as a food additive (436). The essential oil of sassafras is about 75% safrole (437). Sassafras root bark continues to be freely available in health food stores despite evidence indicating its carcinogenicity, and despite legal restrictions prohibiting the use of safrole in foods. Safrole is hepatocarcinogenic in rats and mice and is a major constituent of the oil of sassafras root bark (438).

E. Bay Leaf At least one herb, bay leaf, exhibits some biological effects because of its physical size and shape. Bay leafcan become physically stuck in the pharyngeal pouch (439), becoming

Beier and Nigg

72

Table 9 Natural Chemicals in the Herb Yarrow (Achillea millefolium) and Their Biological Activities

Chemical Achilleine Achilletin Apigenin Atulene Betaine Borneol Bornyl acetate 6-Cadinene Caffeic acid Camphene Camphor

Caryophyllene Chamazulene

Concentration (P94.I dry weight)

0-1 40 255 210 8

Humulene lsoartemisia ketone Limonene Luteolin

Insectifuge Antitumor, choleretic, hepatotropic

1779

(LDLoa 990 mg ipr in rats) analgesic, anesthetic, antiseptic, antipruritic, carminative, deliriant, emetic, rubefacient, stimulant

159

959

Essential oil Eugenol

Hemostat Hemocoagulant Antihistaminic, antispasmodic Antiinflammatory, antipyretic Antimyoatrophic, emmenagogue

602

Choline

Copaene Coumarins Cuminaldehyde p-Cymene

Activity

Anodyne, antiinflammatory, antiseptic, antispasmodic (LDm400 mg iprin rats) lipotropic, hypotensive (LDm2480,mg orallyin rats) antibronchitic, antilaryngitic, antipharyngitic, antirhinitic, expectorant, insectifuge

59 11 369

Anticoagulant (LDS0 1390 mg orally in rats) (LDm4750 mg orallyin rats) fungitoxic, insectifuge

1000-1 4,000 (LDm3000 mg orallyin mice) analgesic, anesthetic, antiseptic, fungicide, larvicide

22 860

171 Antiinflammatory, antispasmodic, antitussive

Naturally Occurring Toxic Chemicals in Foods

73

Table 9 Continued

Concentration (&I dry weight)

Activity Chemical

(LDW3180 mg orallyin rats) analgesic, anesthetic, counterirritant, antipruritic

Menthol Myrcene a-Pinene

22 941

&Pinene Quercetin

713

Rutin

Sabinene Salicylic acid

Ailelochemic, beetle-attractant,expectorant Expectorant, insectifuge (LDm 161 mg/kg orallyIn rats) antiinflammatory, antispasmodic (LOW950 mg/kg ivnin mice) antiatherogenic, antiedemic, antiinflammatory, antithrombogenic, hypotensive, spasmolytic, vasopressor

1235

0-Sitosterol

Stachydrine Tannins a-Terpinene 7-Terpinene Terpinen-4-ol

131 371 431

Terpinolene Thujone abortifacient

48

Tricyclene Triganelline

27

(LDW891 mg/kg body weight orally in rats) analgesic, antipyretic, antirheumatic Antihypercholesterolemic, antiprostatitic, antiprostatadenomic, antitumor, aphrodisiac, estrogenic Cardiotonic Antidiarrhetic, bactericide, viricide Insectifuge

Antiallergenic, antiasthmatic, antiseptic, antitussive, bactericide, expectorant, fungicide, insectifuge (LDLo 120 mg/kg body weight ipr in rats) (LDLo 5000 mglkg body subcutaneously in rats) hypoglycemic

-~~

~~~

"DLo, lowest dose proven lethal in experimental animals. Source: From Refs. 433 and 434.

lodged in the mucous membrane, and blocking the esophagus. It also can rupture Meckel's diverticulum, resulting in severe rectal pain (440). Panzer suggests that bay leaf complications may be a more important source of morbidity than the present literature suggests. Perioric dermatitis can be caused by bay leaf, marjoram, and cinnamon; its cause and pathogenesis are not known (441).

Beier and Nigg

74

Table 10 Compounds in Bishop's Weed (Amnzi mujus L.) Seed

Compound Alloimperatorin Ammirin' Bergapten Graveoloneb Heraclenin Herniarin' lsoimperatorin' lsopimpinellin' Marmesin Marmesinin Oxypeucedanin Oxypeucedanin hydrate Pabulenol Saxalin Umbelliferonea Umbelliferone-(3'-hydroxymethyl-1 t.-buten-l '-yl)-ether' Umbelliferone-(3'-methyl-buta-lt.3-dien-1 '-yl)-ether' Xanthotoxin Total psoralens

1 400-31 00 100-8000

< 100 2300 400-3300 100-1 450 3000

c 100

LYSERGIC ACID AMIDE

H Figure 2 Ergotalkaloids:lysergicacid.

H simple lysergicacidamides.

Porter

658

TRICYCLIC PEPTIDE PORTION 9, IO-ERGOLENE RING

9

A

/

BASIC ERGOPEPTINEALKALOID

Figure 3 Ergotalkaloids:

ergopeptine.

Another class of ergot alkaloids (i.e., ergobalansine; Fig. 6) has been isolated from cultures of Bnlnrlsin spp. (57j; these alkaloids are devoid of the proline moiety in the tricyclic peptide portion of the molecule (compare Fig. 3 vs. Fig. 5). Predicated on the biochemistry for the ergopeptines (14, 31, X), it would appear that two amino acids (e.g.. instead of three) are incorporated in the biosynthesis of this portion of the compound. The toxicity of this alkaloid has not been investigated, but because of the evolutionary relationship between Bnlarzsicl and Clcrviceps (34, 46, 50, 51), future studies will define if ergobalansine is prototypical or a prelude to a distinct class of ergot alkaloids analogous to the ergopeptines.

open

0

H‘ ERGOVALAM: R1=-Me; R2=-iso-Pr

Figure 4 Ergopeptam alkaloids.

Analytical Methodology for Mycotoxins

659

Table 1 General Structure of the Ergot Peptide Alkaloids and Their Major Ions, Atomic Mass Units (amu), Resulting from Electron Impact (ET) (fragments A, B, C) and/or Isobutane Chemical Ionization (CI) (fragments AH, BH, and CH) Mass Spectrometry (see text for explanation) alkaloids Ergopeptine Ergotamine group (R, = -CH,) Ergotamine Ergosine beta-Ergosine Ergovaline Ergobine Ergoxine group (R, = -C,HS) Ergostine Ergoptine beta-Ergoptine Ergonine Ergobutine Ergotoxine group (R, = -i-Pr) Ergocristine alpha-Ergocryptine beta-Ergocryptine Ergocornine Ergobutyrine

R2

MW

CH AH

BH

-CH?Ph -i-Bu -sec-Bu -i-Pr -Et R2 -CH:Ph -i-Bu -sec-Bu -i-Pr -Et R? -CH2Ph -i-Bu -sec-Bu -i-Pr -Et

581 547 547 533 519

268 268 268 268 268

315 28 1 28 1 267 252

245 21 1 21 1 197 183

595 56 1 56 1 547 533

268 268 268 268 268

329 295 295 28 1 267

245 21 1 21 1 197 183

609 575 575 561 547

268 268 268 268 268

343 309 309 295 28 1

245 21 1 21 1 197 183

MW = molecular weight.

CH3

H’

H’

FESTUCLAVINE

PYROCLAVINE

H’ DMYDROELYMOCLAVINE

Figure 5 Festuclavine,pyroclavine,dihydroelymoclavinevia C. ufiicnnu.

Porter

660

Figure 6 Ergobalansine.

The ergopeptines, simple lysergic acid amides, and clavine alkaloids (Figs. 1-3) have been isolated from Neotyphodiuminfected tall fescue (58-62) and cultures of this fungus (63, 64), which is consistent with its evolutionary relationship with Clcrviceps (34, 46, 50, 51). Although ergovaline is the major ergopeptine alkaloid in N. coenoylzicdurninfected tall fescue (58-60,65), lysergic acid amide (or ergine; Fig. 2) can exist in concentrations approximately equal to that of ergovaline (66). Lysergic acid amide is now accepted as a true natural product, but it can, as well, result from the solvolytic cleavage of lysergylmethylcarbinolamide (Fig. 7 ) (31, 67). This is a classic example for compound reaction and/or stability after extraction from its natural matrix. Ergonovine (Fig. 2), another simple lysergic acid amide, also has been isolated from endophyte-infected fescue seeds (61), but this compound may be from CZnviceps contamination of the seeds (59) and further serves as an example of howsecondary infection (or Contamination) ofsamples

HLYSERGYLMETHYLCARBINOLAMIDE

LYSERGIC ACID AMIDE

Figure 7 Lysergic acidmethylcarbinolamide slovolytic cleavage to lysergic acid amide.

Analytical Methodology for Mycotoxins

661

can obscure analytical results. Because ergonovine is found in conjunction with the ergopeptine alkaloids in Claviceps-infected wheatand rye (68), it is possible this alkaloid may also be present in endophyte-infected grasses. The clavine alkaloids chanoclavine(s), penniclavine, elymoclavine, and agroclavine (Fig. 1) also have been isolated from endophyte-infected tall fescue (60) and are precursors in the biosynthesis of the simple lysergic acid amides and the ergopeptines (14, 31, 32, 69). 2. Isolation and Identification Since the endophytic fungi of pasture grasses are systemic infections of their host versus the localize seed head infection of Clmiceps (34, 41, 42), the extraction of the ergot alkaloids from the two matrices illustrates how background materials influence the isolation and analytical processes. There are a variety of procedures for their extraction and isolation from endophyte-infected pasture grasses (i.e., tall fescue, perennial ryegrass, etc.), which represent variations on those employed for Clnviceps-infected grains (70,71). Current methods of choice involve extraction with either an aqueous tartaric or lactic acid solution; lactic acid appears to work best for the extraction of ergovaline from the infected fescue (72), partition chromatography with chloroform or methylene chloride at an appropriate pH, column cleanup procedures using either silica, alumina, or an ion exchange resin, and identification and analysis using cochromatography (TLC and/or HPLC) with ultraviolet or fluorescence detection. Mass spectrometry (MS) also has been employed for the identification and quantitative analysis of the ergot alkaloids. The ergot alkaloids are extremely susceptible to photolytic and air oxidation, hydration, and epimerization at the C-8 position of the ergolene ring (14, 32, 69) (Fig. 3). Epimerization of the C-8 position occurs in either acidic or basic conditions, and therefore, the isolation of the C-8 epimers (designated with the suffixal-inine, i.e., ergovaline vs. ergovalinine) occurs in most extraction procedures. Decomposition and epimerization may be minimized by working under subdued or yellow light, by concentrating extracts in vacuo at room temperature or less (i.e., 125"C), and by concentrating small volumes of extracts under a stream of nitrogen. Storing dried concentrates in amber vials under nitrogen at or below 0°C will help prevent further decomposition. Moubarak et al. (73) have successfully stored ergovaline at -4°C for up to 12 months. Furthermore, ergovaline decomposes rapidly when extracted from non-freeze-dried plant tissues (69). Thus, observing the correct precautions during sample collection, handling, and preparation is crucial for the isolation and quantitative analysis of the ergot alkaloids from Claviceps-infected grains and/or endophyte-infected grasses. The total concentration of ergot alkaloids in endophyte-infected tall fescue varies with the season and amount of nitrogen fertilization (60,65,74-76). Whether the relative concentration of the individual ergot alkaloids varies with these parameters is currently unknown and such factors should be considered (and recorded) prior to sample collection and analysis. 3. High-Performance Liquid Chromatography HPLC with either ultraviolet or fluorescence detection is the preferred method for routine screening and analysis of the ergopeptine alkaloids in endophyte-infected grasses (55, 59, 65, 66, 72, 73, 77-79). A rapid, simplified HPLC method for the analysis of the ergot alkaloids associated with N. coerzophialum-infected tall fescue has been developed (66). Extraction of infected seed or grass with alkaline methanol, followed by filtration, and direct HPLC analysis (fluorescence detection) with a mobile phase of either 60 or 70% alkaline methanol results in separation of ergovaline, ergovalinine, lysergic acid amide,

662

Porter

and its isomer isolysergic acid amide (erginine). The use of gradient elution allows for the complete separation of complex mixtures of the ergot alkaloids (66). A unique preparative method for the isolation and purification of large quantities of ergovaline has been reported (73). This procedure involves a modification of previous methods (65, 72, 80) in which the infected seeds are extracted with a 5% aqueous lactic acid solution and the ergot alkaloids are adsorbed onto SM-2 Biobeads (BioRad, Hercules, CA). Extraction of the Biobeads with methanol, followed by an HPLC cleanup procedure using a C- 18 RP column (Vydac, Separations Group, Hesperia, CAI, and HPLC analysis using gradient elution with acetonitri1e:ammonium carbonate:methanol as the mobile phase results in pure ergovaline (295%). Zhang et al. (79) have employed an amberlite XAD-2 exchange resin for a rapid cleanup step after extracting infected fescue seed with a 5% lactic acid: methanol (4: lv/v) solution. Other HPLC methods (81) have been employed for the analysis of ergovaline/ergovalinine distribution in the leaves of fescue colonized by two different Neofhyphodium spp. Scott et al. (68) and Rottinghaus et al. (82) have reported additional extraction, cleanup, and HPLC procedures for the analysis of the ergopeptine alkaloids in cereal grains, flour, and feeds. 4. Thin-Layer Chromatography After the ergot alkaloids have been extracted into a suitable organic solvent, TLC on silica gel remains one of the most powerful tools for the analysis and identification of these compounds. The major advantages of TLC are that several samples may be analyzed at the same time, TLC does not involve expensive instrumentation, relatively small quantities of standards may be used, mg quantities of the individual alkaloids may be separated and purified with preparative TLC, and it compliments MS in the confirmatory identification and analysis of epimeric alkaloid mixtures (see below). Perellino et al. (54) have reported a TLC procedure on silica gel for the separation of most all of the known ergot alkaloids. When the TLC plates are developed in methylene chloride: isopropyl alcohol (92 :8 v/v) three times (drying the plates between ~uns),these alkaloids separate into the isolysergic acid group (i.e., -inirze epimers), the ergotoxine group, the ergoxine group, and a mixture of the ergotamine and clavine groups. Removal of the silica from the area of the plates consistent withthe known standards, extraction of the alkaloids from the silica using methanol: chloroform (1 :4 or 1 : 1 v/v) (54, 71), and rechromatography in chloroform: methanol (9 : 1 or 4 : 1 v/v) separates ergovaline from the clavine alkaloids (agroclavine and chanoclavines) (63). Other solvent systems used for the TLC analysis of these compounds are listed in Table 2. Table 2 Solvent Systems3 Effectively Used for TLC on Silica Gel for the Separation and Identification of Ergot Alkaloids CH2C12:iso-PrOH (92: 8; 90 : 10; 75 :25) CHC17:MeOH (95:5; 90 : 10; 80 : 20) CHC&:MeOH (90: 10) in a saturated NH3 atmosphere CHC13:MeOH :NH3 (94 : 5 : 1) CHC13 :Et,NH (90 10) Benzene : dimethylformamide (86 : 5 : 13: 5 ) ' All systems (v/v) are in a saturated atmosphere. in tanks lined with Whatman No. 1 filter paper. 54. 63, 64, and 7 1.

Sowce: Refs. 21,

Analytical Methodology for Mycotoxins

663

Visualization of the ergot alkaloids on a TLC plate may be accomplished with a hand-held UV light at 254 and 366 nm. The 9,lO-double bond in the wring of the ergolene portion of the molecule (Fig. 1) is conjugated with the indole nucleus and gives the ergopeptine alkaloids their characteristic bright, pale blue fluorescence (UV lambda max in methanol at approximately 315 and 242 nm). Those ergot alkaloids devoid of the 9,lOdouble bond give a characteristic dark blue absorption under 254 nm (UV lambda max in methanol at approximately 292,280, 275, and 222 nm) indicative of the indole nucleus. Spraying the TLC plates with a solution of p-N,N-dimethylaminobenzaldehyde(van Urk's reagent; 83) followed by spraying with a 1% sodium nitrite solution (water: ethanol, 1 : 1 v/v; 84) produces intense blue spots that are also characteristic of the ergot alkaloids. Colorimetric analysis at 590 nm of a crude alkaloid fraction relative to a known standard (i.e., ergonovine maleate; ergotamine tartrate) provides a method for quantitating total ergot alkaloids in crude extracts (85). Individual ergot alkaloids may then be identified and quantified by a combination of TLC, HPLC, and/or MS. 5. Mass Spectrometry Identification and quantitative analysis of the ergot alkaloids isolated from infected grains, grass, and laboratory cultures using MS include electron impact (ET) (63), chemical ionization (CI) (64, 86), and tandem mass (MS/MS) spectrometry (58, 60, 62, 70). Under lowresolution electron impact (70 eV), the ergopeptine alkaloids pyrolytically decompose into the lysergic acid amide, the cyclic peptide, and diketopiperazine fragments A, B, and C, respectively (Fig. 8). These fragments then undergo E1 and produce spectra characteristic of the ergolene ring and the peptide portion of the parent molecule. Fragments useful in the interpretation of these spectra occur at m/z+ 70, m/z+ 125, and m/z+ 154 atomic mass units (amu), which are characteristic of the proline moiety (63, 87). These ions, in combination with the lysergic acid amide ion at m/zf 267 amu (ion A, Fig. S), are indicative of an ergopeptine alkaloid. Using ergovaline for example, in addition to m/z+ 267 amu, the other two major fragments associated with ions B and C (i.e., fragments indicative of the methyl substituent at R1 and the isopropyl substituent at R2: Figs. 3 and 8; Table

Figure 8 Mechanism for the mass fragmentation of the erogopeptine allkaloids (EI; CI).

664

Porter

1) occur at m/z+ 266 and m/z+ 196 amu, respectively. The major disadvantage with low resolution EIMS (70 eV)in the analysis of the ergopeptine alkaloids is the low abundance (i.e., 5 1%)of the molecular ion and the diagnostic fragment ion B when R2 is an alkyl substituent (i.e.. ethyl-, n-butyl-, isobutyl-, sec-propyl-, isopropyl-; Figs. 3 and 8; Table 1). Although fragments associated with theergolene nucleus are isobaric with those fragments related to the cyclic peptide (B) and diketopiperzine (C) portions of the parent molecule (64), ergovaline and its C-8 epimer ergovalinine produce a high abundance of ion m/z+ 196 amu (ion C) (63). Spectral interpretation of complex mixtures of the ergopeptines, however, becomes somewhat more difficult. Isobutane chemical ionization mass spectrometry (CIMS) has been employed in the identification of the ergopeptine alkaloids (64, 86). This method involves an ion-molecule reaction of the alkaloids in the presence of a reagent gas (isobutane). The ion-molecule reaction results in reduced fragmentations, as seen with low-resolution EIMS, produces simplified spectra, and thus circumvents interpretation difficulties of the more complex spectra and those resulting from mixtures of these alkaloids. Under isobutane CIMS, the lysergic acid amide (A), cyclic peptide (B), and diketopiperazine (C) molecules (Fig. 8) abstract a proton from a r-butyl cation (which is generated in the mass spectrometer) and the resulting ion-molecule reaction produces spectra containing three major ions represented by AH, BH, and CH in Fig. 8 and Table 1. These ions are 1+ amu greater than the parent fragments A, B, and C (58, 62, 64, 86). The major fragments used to identify most of the known ergopeptine alkaloids by this method are outlined in Table 1. Although ergovaline and its C-8 epimer ergovalinine produce the same three major fragments at m/z+ 268 (AH), m/z+ 267 (BH), and m/z+ 197 (CH) amu and cannot be distinguished by this method, both compounds (as with the other ergopeptines and their C-8 epimers) separate nicely using TLC and/or HPLC analysis (54, 59, 63, 65, 71, 82). Tandem mass spectrometry (62) also has been employed in the separation and analysis of ergopeptine alkaloids from both endophyte-infected tall fescue and perennial ryegrass ( 5 8 , 75, 88). In an overly simplified discription, MWMS (which also is conducted in the presence of a reagent and/or a target gas) uses one stage of mass separation to isolate the individual alkaloids of interest in a crude extract; depending on the ionization mode, this usually involves the molecular ion, a protonated molecular ion, or a molecular anion; a second stage of mass separation is then employed to analyze the product and/or daughter ions (also dependent on the reagent and/or target gas). The fragmentation mechanisms for the ergopeptine alkaloids utilizing MS/MS with isobutane as the reagent gas and argon as the target gas are analogous to that described for isobutane CIMS and are described i n detail (62). Major advantages of this system are that complex mixtures of these compounds can be analyzed without prior cleanup, only small samples of extracted material are needed, and isomeric species (i.e., ergosine vs. beta-ergosine; Fig. 3; Table 1) can be distinguished. However. MS/MS analysis requires expensive instrumentation not readily available to most laboratories, and like CIMS, this system cannot distinguish between epimeric ergopeptine alkaloids. Subsequently, MS/MS is not practical for routine screening of toxic or infected grasses. Field desorption mass spectrometry also has been used in the identification of the ergopeptine alkaloids (87) and provides extremely simplified spectra along with intense molecular ions. Recently, Porter et al. (21, 56) have used a combination of TLC and GUMS to analyze and quantitate the clavine alkaloids (dihydroelymoclavine, festuclavine, and pyroclavine; Fig. 5) extracted from C. afi-icann-infected sorghum. Infected sorghum was extracted with MeOH-H,O (70: 30 v/v) containing 1% NH40H at room temperature; the

Analytical Methodology for Mycotoxins

665

aqueous solution was extracted with CHCll and the crude alkaloid material subjected to preparative TLC (silica gel) using CHC13-MeOH (9: 1 v/v) followed by CHC13-MeOH (95 :5 v/v) as the developing solvents. Fractions consistent with festuclavine and pyroclavine, dihydroelymoclavine, chanoclavine I, and dihydroergosine (i.e., Rf, visualized under UV 254; and giving a blue reaction to p-dimethylaminobenzaldehyde) were collected, eluted from the silica (CHC13-MeOH, 1: 1 v h ) , and the eluate concentrated to dryness (N2).Each alkaloid fraction was subjected to GUMS analysis neat and also after reaction with MSTFA. Festuclavine and pyroclavine could be analyzed neat (M' 240) and as their TMS-derivatives (M' 312; TMS-moiety attached to the indole-N; Fig. 9), whereas chanoclavine and dihydroelymoclavine were analyzed as their TMS-derivatives (M' 472 and M+ 400, respectively) (Figs. 9 and 10). With MSTFA, chanoclavine I gave a mixture of the di-TMS-(M' 400) and tri-TMS-derivatives (M+ 472) (Fig. 10); this mixture was quantitatively converted to a single compound by reaction with MBTFA, in which the substituent at position 6 [i.e., the CH3-N-H (disubstituted derivative) and the CH3-N-TMS (trisubstituted derivative)] was exchanged for CH,-N-COCF, (M+ 496) (cf. Fig. 10). The major amu(s) and fragmentation patterns for all TMS derivatives follows the analogous patterns as that for the parent alkaloid (EIMS, 70 eV), with the expected shift of 73 amu for each displaceable proton. Subsequent G U M S evaluation of costaclavine and agroclavine [neat (M+ 240 and M' 238) and as their TMS derivatives (M' 3 12 and M+ 3 lo), respectively], lysergol-TMS (M+ 398), elymoclavine-TMS (M' 398), and dihydrolysergamide-TMS (ndz' 324, M+-NH7)proved this method unequivocal for the identification and quantitation of this class of ergot alkaloids in the nanogram range using both SCAN and SIM modes. Dihydroergosine was analyzed using both TLC and HPLC.

6. Loline Alkaloids The loline alkaloids (Fig. 11) in N. coenophinlurn-infected tall fescue are produced by the fungus as a plant host-defense mechanism in response to insect herbivory and, except as a deterrent to insects, may not fall under the classic definition for mycotoxins (see

$ *

H

CH,

N T/MS

TMS

TMS-ESTUCI>AVINE

TMS-PYROCLAVINE

TMS TMS-DIHYDROELYMOCLAVINE

Figure 9 TMS-festuclavine, -pyroclavine, -dihydroelymoclavine alkaloids.

Porter

666

($, CH2OH

H3

MSTFA

/

N

CH2OTMS CH7OTMS

/

m/z 256

( f

H' CH20TMS

MBTFA

N TMS TMS

m / z 400

& + +

S)CH,

/

N

TMS

dz472

ThS

Figure 10 TMS-chanoclavine I alkaloid.

above). This defense mechanism may involve interactions between both the endophyte and its host grass and, as with the ergot alkaloids, suggest a true symbiosis between the endophytes and their plant host (50, 89-91). Moreover, Petroski et al. (92) have suggested the lolines may be alleopathic, thus improving the ability of the infected grass to compete with other grasses. The chemistry, occurrence, and biological effects of the loline alkaloids and associated endophyte-grass interactions have been reviewed (89, 93). Capillary gas chromatography using either FID and/or MS detection is an established method for the analysis of the loline alkaloids (89, 92, 94). Extraction of ground seed (approximately 5 g) with methylene chloride :methanol :ammonia (75 :25 :0.5 v/v/v),

LOLINE

RI

R2

CH3

H

N-ACETYLLOLINE COCH3 CH3 N-FORMYLLOLINE CHOCH3 N-ACETYLNORLOLINE COCH3 H N-METHYLLOLINE CH3

Figure 11 Lolinealkaloids.

cH3

Analytical Methodology for Mycotoxins

667

followed by filtration, provides extracts ready for GC/FID or GUMS analysis. Forage samples may be extracted analogously, but prior to analysis, a sulfonic acid solid phase cleanup step of the forage extracts is necessary to remove substances that interfere with the assay. The detection limit for N-acetylloline and N-formylloine is 10 ng. Tepaske et al. (94) have described a similar GUMS procedure for the analysis of the loline alkaloids in bovine urine and plasma. The loline alkaloids do not provide a well-defined molecular ion in the mass spectrum (70 eV), but their separation under GC conditions and characteristic mass fragmentation patterns (89) allow for their unequivocal identification and quantification. Petroski et al. (92) and Powell and Petroski (89) reported a method for the separation of the loline and the ergot alkaloids from endophyte-infected tall fescue whereby an alkaloidal extract is subjected to column chromatography using Sephadex LH-20. The loline alkaloids pass through the column and the ergot alkaloids are recovered by exhaustive elution of the LH-20 with methanol. Also, countercurrent chromatography for the separation of milligram quantities of N-methylloline, N-acetylloline, and N-formylloline (Fig. 11) from an endophyte-infected tall fescue seed has been reported (61). Total lolines (defined as N-formyl- and N-acetylloline) occurring in endophyte-infected tall fescue seed (3263 pg/g) and forage (1723 pg/g) were quantified using GC/FID (90). Concentrations of these alkaloids (as with the ergot alkaloids) in forages vary with season, the amount of nitrogen fertilization, grazing pressure, and the amount of insect herbivory (93,95, 96). 7. Paxilline and the Lolitrem Alkaloids from N. lolii-Infected Perennial Ryegrass Evidence suggests that the indole-isoprenoid lolitrems are almost as diverse as the ergot alkaloids. Paxilline and lolitrem B (Fig. 12) are the two major alkaloids associated with perennial ryegrass staggers in sheep, cattle, and other livestock (97-101). Studies involved with determining the biosynthesis of paxilline and lolitrem B resulted in the identification of alpha-paxitriol, lolitriol, and the lolitrems A, C, D, and E (102).These additional minor lolitrems may contribute to the overall toxicity of paxilline and lolitrem B. H

PAXILLINE

LOLITREM B

Figure 12 Paxilline and lolitrem B.

Porter

668

Gallagher et al. (97) reported an isolation and screening method for lolitrem B in N. Mi-infected perennial ryegrass. One gram of oven-dried, milled grass was extracted with chloroform: methanol (50 ml; 2 : 1 v/v) for 1 hr. One milliliter of the extract was dried under nitrogen, reconstituted in methylene chloride (2 ml), and subjected to a cleanup step on silica. The eluent (100 pl)from the silica was then analyzed by HPLC on a Zorbax Silica column (DuPont, Wilmington, DE), using methylene chloride: acetonitrile (80 :20 v/v) as the mobile phase. Recovery of lolitrem B was 93-97% with detection limits at 500 pg (fluorescence detection). This HPLC method has been used to screen up to 80 samples per day. The amount of lolitretn B in the infected grass necessary to elicit the staggers syndrome is only 5 ppm.

8. Peramine Although peramine (Fig. 13) is a plant metabolite and the major insect deterrent in N. coelzophiaIlllll-infected tall fescue and N. lolii-infected perennial ryegrass (91, 103-106), its isolation is mentioned to exemplify a unique separation method for both peramine from lolitrem B using a two-phase extraction system. Freeze-dried, ground grass (100 mg) is first extracted with methano1:chloroform (3 ml) and followed by concurrent extractions with hexane and water (3 ml each). The aqueous and organic phases are separated by centrifugation. Lolitrem B was analyzed in the organic phase as previously reported (97), whereas peramine, after minor cleanup using ion-exchange chromatography, was analyzed in the aqueous phase by HPLC with a mobile phase of acetonitri1e:guanidinium formate (pH = 3.7). Recovery of peramine is 93-loo%, with detection limits at 1 yg/g of infected grass. Alternatively, peramine may be analyzed in the aqueous phase, after an ion-exchange cleanup step with two minicolumns connected in series. The first column (BioRad AG 2 X 8, 200-400 mesh) is in the hydroxide form and the second column (Analytichem Bond Elut CBA) is in the carboxylic acid form. After the aqueous phase is aspirated onto the columns, they are washed with 80% aqueous methanol (3 ml), the columns separated, and peramine eluted from ihe acid column with aqueous methanol and formic acid. Pera-

PERAMINE

Figure 13 Peramine.

Analytical Methodology for Mycotoxins

669

mine is then analyzed by TLC using chloroform :methanol :acetic acid :water (30 : 10 : 1 : 1 v/v/v/v) as the developing solvent (103). A rapid, sensitive, reverse-phase TLC method for the analysis and quantification of peramine in crude extracts of several endophyteinfected grasses has been reported (107) and the characteristic low-resolution mass fragmentation (70 eV) of peramine has been defined (106).

B. Fusarium Metabolites Another mycotoxin problem has been recognized throughout the world since: (1) F. rnoniliforme and the fumonisins (Fig. 14) were associated with equine leukoencephalomalacia (108-1 10); (2)the fumonisins were statistically associated with esophageal cancer in humans in certain areas dependant on corn as the staple diet (12, l l 1-1 17); and (3) the routine occurrence of the fumonisins and other Ftrsa~iun1toxins in corn, wheat, barley, rice, and other cereal grains (1 18- 121). Although Fz4sarium species and their toxic metabolites occur most prevalently in corn, wheat, and barley, they are also found in nuts, fruits, and vegetables, and in other nonfood items of economic importance (e.g., tobacco, cotton, forage grasses, alfalfa, red clover, flax) (119, 122). Corn, wheat, and barley comprise twothirds of the world cereal production and the numerous Fusarium species associated with animal and human health problems ( 1 3 , 124) warrant the most expedient and precise analytical procedures for their toxic metabolites. 1. Fumonisins The ubiquitous detection of the fumonisins in cereal grains and especially corn, rice, and corn and rice-based products indicates that the potential for human and animal exposure is a worldwide problem (6, 12, 109, 116- 120, 125). Several fumonisins have been identified from F. morzilifomle-infected grains and defined as fumonisin B, , B2, B3, B4, AB,, and AB2 (109, 120, 121). Fumonisin B, is the major metabolite found in nature and also

ACID

FUSARIC MONILIFORMIN

OH 0 I

ZEARALENONE

OH

OH

OH

DEOXYNIVALENOL

Figure 14 Fz4sarizlnz mycotoxins: moniliformin, fumonisin B,, fusaric acid, zearalanone. deoxynevalenol.

670

Porter

in cultures and defined as the 2-amino-12,16-dimethyl-3,5,10,14,15-pentahydroxyicosane with a propane-l,2,3-tricarboxylatesubstituent at C-14 and C-15 positions (109, 120). Fumonisins B, and B, are the C10 and C5 deoxy analogs, respectively, whereas B4 is missing the hydroxyl moiety at both C-5 and C- 10. Fumonisin AB and AB2are the corresponding N-acetyl analogs of B1 and B?, respectively. Since their isolation and identification, numerous HPLC procedural variations have been reported (120-121, 125-135). Briefly, the fumonisins may be extracted with either methanol-water or acetonitrile-water followed by cleanup on an anion-exchange resin. The resin is then eluted with acetic acid-methanol, followed by derivatization of the extract with o-phthalaldehyde (OPA), and HPLC analysis with fluorescence detection (125). Variations of both derivatization (i.e., 9-fluorenylmethylchloroformate and 4-fluoro-7-nitrobenzofurazan (129, 133) and HPLC analysis (isocratic and gradient elution) have been employed (125-135). An improved preparative HPLC procedure for funonisin BI (B? and B3) (underivatized; using light-scattering detection) and the subsequent quantitative analysis have also been reported (127, 128); fumonisin B, purity was 195% with a recovery of >90%. Other preparative procedures using amberlite, XAD-2, silica gel, and CI8reverse phase-chromatography have been reported (136, 137). Meredith et al. (127, 128, 138) have further utilized centrifugal spinning TLC and analytical HPLC with fast-atom bombardment mass spectrometry for the preparative and quantitative analysis of the fumonisins, and Rottinghaus et al. (139) have reported a sensitive TLC method for the detection of fumonisin B, and B2. In studies directed at the removal of fumonisins from contaminated corn, Sydenham et al. (13 1) have reported the analysis of partially hydrolyzed fumonisin B I using a combination of column, thin-layer, and HPLC-electrospray MS to isolate, purify, and quantitate the corresponding aminopentol of this mycotoxin. Procedural analysis for another analog of fumonisin B, (i.e., the N-acetyl-C-15-keto-derivative)isolated from corn cultures of F. yrol(femtznzr has been reported (140), and Sydenham et al. (130) have compared a monoclonal antibody-based competitive direct enzyme-linked immunosorbent assay (CDELISA) with the HPLC determination of fumonisin in corn. The structurally related fumonisin-like compounds present in naturally contaminated corn may contribute to the differences between these two methods (i.e., CD-ELISA results were consistently higher than the HPLC results) and emphazises the importance of specific chemical analyses for the mycotoxins in question. The CD-ELISA however may still be used as an initial semiquantitative screening technique (130). Most recently, Meredith has reported a compendium on the isolation and characterization of the fumonisins and the reader is referred to this chapter (141). Then too, the co-occurrence and analysis of the fumonisins with other F~sm-irnntoxins (Le., zearalenone, deoyxnivalenol, and the other trichothecens) are referenced below.

2. Zearalenone Zearalenone (ZEN) and related metabolites are nonsteroidal estrogenic mycotoxins produced by several species of Fz~sariun~ (10, 142) that are routinely surveyed in cereal products (143) and directly related to hyperestrogenism and infertility in swine (10, 142) and precocious puberty in children (144). Bennett et al. (143) developed an HPLC method using UV or fluorescence detection with an average recovery of 82-100% for ZEN and its metabolite a-zearalenol. Doko et al. (125) employed an improved HPLC method in conjunction with TLC for the analyses of ZEN and the fumonisins in cereals and cerealbased foods from Africa, and Ryu et al. (145) have described a GUMS analytical method

Analytical Methodology for Mycotoxins

677

for the simultaneous detection and quantitation of naturally occurring ZEN and six major trichothecenes in barley and maize (see below). 3. Trichothecenes and Co-occurrence with Other Fungal Toxins Over 100 trichothecenes have been identified from Fusarium and other genera (Myrothecium, Trichothecium, Cephulosporiurn, Verticirnonsporium, and Stachybotqs) (145), but major analytical interests have concentrated on those of Type A and B (6) because of their diverse toxicological effects in both animal and human disorders and their routine occurrence in cereal grains and stored grain products ( 1-3,6, 10- 12, 145- 153). Although deoxynivalenol (DON; vomitoxin) is only one of several tricothecene mycotoxins produced by Fuscrrium spp. (F. nlorziliforrne, F. C14~?11O~l411Z, F. graminenrum, F. roseum, etc.), it is among the most frequent tricothecenes analyzed in cereal crops (10, 12). DON is a 12,13-epoxytricothecene associated with feed refusal (i.e., reduced weight gains and growth depression) and the emetic responses primarily in swine (10, 122, 154). Current food safety guideline recommendations for this mycotoxin in cereals used for humans is 2 ppm with no more than 1 ppm for foods used in infant formulations (1 1). Other structurally related tricothecenes routinely screened in cereals are 3-acetyldeoxynivalenol (3ADON), 15-acetyldeoxynivalenol (15-ADON), 3,15-diacetyl-deoxynivalenol (3,15DADON), nivalenol (NIV), 4-acetylnivalenol (4-ANIV), 4,15-diacetyl-nivalenol (4,15DANIV), Fusarenon-X (F-X), T-2 toxin (T2). and HT-2 Toxin (HT-2) (145-150). Ryu et al. (145) have developed a GUMS procedure for the analysis of DON, 3ADON, NTV, F-X, T-2, HT-2, and ZEN in cereal grains that involves extraction of the pulverized cereal samples with actonitrile-water, defatted with n-hexane, followed by solid-phase extraction using a florisil column. After elution from the column with chloroform: methanol (9 : 1). the mycotoxins were derivatized with N,O-bis(trimethyl)acetamide/ trimethylchlorosilane and analyzed by GC (EC and/or MS) with a mean recovery of 913 % for DON, 3-Ac-DON, F-X, T-2 toxin, HT-2 toxin, NIV, and ZEN. Previous investigations (147) compared the extraction efficiency and quantitative recovery of DON and NIV from florisil and sep-pak-silica cartridge columns and their confirmation by GC-EC and GCMS analyses; the range of recovery for the two mycotoxins was 89-99% for DON and 23-99% for NIV. These investigators emphasize the critical points necessary for high recovery of the toxins with solvent selection for extraction, subsequent cleanup procedures, and also recovery of the mycotoxins from the extracts and the different commodities (i.e., wheat, barley, corn, etc.). Other studies (146) have outlined the GUMS conditions and analysis using selective ion monortoring (SIM) of the trimethylsilyl derivatives of DON, 3-ADON, 15-ADON, 3,15-DADON, NIV, 4-ANIV, and 4,15-DANIV extracted from rice cultures of F. grurninarium isolated from barley and corn. The detection limit of this procedure is reported at ca.10 ppb per mycotoxin. These authors emphasize the utility of the procedure for defining chemotaxonomic types of F. gruminarium (and other Fusnriutrl spp.), the significance of geographic location, and the spectrum of mycotoxins produced by the isolates. Furthermore, this report describes the co-occun-ence of ZEN (via HPLC analysis) by the tricothecene-producing isolates from corn (5 1.4%) and barley (31.3%) along with the regional differences in trichothecene production. Analysis of F. grc/mirzelrium- and F. crookwellense-infected maize by extraction (acetonitrile/water, 82/ 18 v/v), column cleanup (employing charcoal, celite, and aluminium oxide), and TLC analysis further demonstrated the co-occurrence of DON, 3- and 15-ADON, and NIV with ZEN and differentiated between the production of NIV instead of DON when ears are

672

Porter

infected with F. croohvellense; ZEN accumulation occurred in cobs colonized by both species (150, 155, 156). Other Fusnriunz toxins that have provided unique problems with analytical procedures are: the fusarins (157, 158), beauverisin (159), fusaproliferin (160), fusarochromanone (161, 162), fusaric acid (10, 18, 20, 21, 1221, and moniliformin (27, 154, 163). The fusarins are pyrrolopolyketides of which fusarin C is mutagenic (157, 158); beauverisin is a cyclic hexadepsipeptide consisting of three N-methylphenylalanyl- and three 2-hydroxy-3-methylbutyric acid residues in a continuous-alternating sequence and is reported toxic to both mammalian and insect cell lines (159); fusaproliferin is a sesterterpen, toxic to both brine shrimp larvae and human B-lymphocytes cell line (160). Fusarochromanone, 2,2-dimethyl-5-a1nino-6-(3’-an~ino-4’-hydroxybutyryl)-chromo~~e, causes tibial dyschondroplasia in avian species and reduced hatchability in fertilized eggs (161, 162), while fusaric acid (5-butylpicolinic acid) is a common metabolite of several Fuscrrir4rz-l species (F. morzilifonne, F. o q s p o n ~ mF. , subglrltinnns, F. yrol(femtunz, F. solalzi, F. jidjikuroi, F. croolwellense, F. napiforme, F. lnteritium, F. thcrpsiszurn (10, 18, 20, 21, 122). Analysis of fusaric acid and its natural occurrence with other Fusnriunz toxins (i.e., ZEN, fumonisins, DON, other tricothecenes, and ergot alkaloids) has been reported (20, 21). Moniliformin (27, 154, 163) is an unusual cyclobutadione produced by a number of Fuscrri~rm isolates and toxicity data have been reported (27); it too has been isolated in conjunction with DON and ZEN (154). The simultaneous occurrence in foods and feeds of mycotoxins from Fusclriunz, Aspergillus, Pelzicillizm, Alternaria, and Clcrviceps has generated major concerns about the synergistic activity between the toxins and created a unique challenge in the screening, detection, and analysis of these compounds. In addition to the procedures outlined above for the isolation and identification of several analogs within a series of mycotoxins, Rava (148j employed a variety of GC/ECD, TLC, HPLC, and irnmunoassays to investigate the co-occurrence of DON, NIV, T-2 (analyzed by GC/ECD) (1 16, 164,165), ZEN,alternariol monomethyl ether (an Altermrin metabolite), and ochratoxin (a Penicillium toxin) (both analyzed by TLC) (166, 167), along with the fumonisin (analyzed by HPLC) (132) and aflatoxin B I (an Aspergillus metabolite, analyzed by immunoassay) (168). Analogously, Wang et al. (169) demonstrated the simultaneous analysis of NIV, DON, and T-2 (with GUMS), fumonisin BI, B2,and B3 (by HPLC with fluorescence detection), and aflatoxin B, (by ELISA). Frisvad (170) has reported a collective HPLC profile for Penicillium, Aspergillus, and Fusrrriurn polyketides, terpenes, and alkaloids; this procedure represents the separation of 134 secondary fungal metabolites with elution times between 1 and 34 min. For the individual analysis of most of the Pellicilliu~n, Asyergillu,~, and Alternaria toxins (ochratoxin, citrinin, patulin, penicillic acid, roquefortine, cyclopiazonic acid, verrucosidin, aflatoxins, sterigmatocystin, tenuazonic acid, alterariol, etc.), the reader is referred to Scott (4), Smith (5), Abramson (7), Panigrahi (8), Viscinti and Sibilia (9), Cole and Cox (27), Savard and Blackwell (28) and Turner and Aldridge (29).

V. SUMMARYANDCONCLUSIONS In addition to the discovery of mycotoxins in cereal grains and other foods and feeds, cheeses and milk, other dairy products, sausages, fruits and nuts, vegetables, and alcoholic beverages (i.e., beer, wine, and whisky) have all come under surveillance for mycotoxin contamination. Hence, a plethora of rapid, sensitive, and efficient mycotoxin screening

Analytical Methodology for Mycotoxins

673

kits have been developed, but these are only mentioned to give the reader an idea of other techniques available that would suggest mycotoxin contamination. Within the last decade, immunological techniques involving monoclonal and polyclonal antibodies, radioimmunoassay, and ELISA have gained wide acceptance with varying degrees of success depending on cross-reactivity and/or specificity of reactants and analytes, and a detailed prospectus is given in another chapter. The natural occurrence of saprophytic, parasitic, and endophytic plant fungi (both localized and systemic) and their evolutionary processes directed at species survival more than suggest an ecological justification for the production of previously referred to secondary metabolites or mycotoxins. Survival mechanisms (Le., physiological, reproductive, defensive, etc.) among these species, their economic significance to production, and their role in human and animal health underscores the importance of definitive analytical methodology for mycotoxins in our food and feed products. Environmental concerns to reduce the volume of herbicides and fungicides have precipitated a movement toward eliminating these practices and going to a no-till agricultural system. Subsequently, fungal infection and mycotoxin contamination of cereal grains, stored grain products, agricultural commodities, field crops, forages, and pasture grasses is a story without an end. However, with the continued judicious development of new analytical technology, advances in tnycotoxin research (i.e., the chemical isolation and identification and toxicology investigations) should provide avenues for understanding fungal-plant growth interactions and contribute to the developtnent of safer and more nutritious products for a global economy.

REFERENCES 1. Mycotoxic Fungi, Mycotoxins. Mycotoxicoses:An Encycolopedic Handbook. Vol.1. Mycotoxic Fmgi and Chemistry of Mycotoxins. T. D. Wyllie and L. G. Morehouse (Eds.). Marcel Defier, New York. 1977. An EncycolopedicHandbook.Vol. 2. 2. MycotoxicFungi,Mycotoxins.Mycotoxicoses: MJrotoxicoses of Domestic and Lnboratory Animals, Poult~y,and Aqrlatic Invertebrates and Vertebrates. T. D.Wyllie and L. G. Morehouse(Eds.).MarcelDekker. New York. 1977. 3. Mycotoxic Fungi, Mycotoxins, Mycotoxicoses:An Encycolopedic Handbook. Vol.3. Mycotoxicoses of Mail and Plcrnts: Mycotoxin Control and Regulatory Practices. T. D. Wyllie and L. G. Morehouse (Eds.). Marcel Dekker. New York. 1977. 4. Scott, P. M. 1994. Penicillirrr~zand Aspergillus toxins. Ch. 5, pp. 261-285. In: Mycotoxins irz Grain: Cor~~pozcnds Other Tlmn Aflatoxirl. J. D. Miller and H. L. Trenholm (Eds.). Egan Press, St. Paul, MN. 5. Smith. J. E. 1997. Aflatoxins. Ch. 19. pp. 269-285. In: Handbook of Plant and Furrgal Toxicants. J. P. F. D’Mello (Ed.). CRC Press, Boca Raton. FL. 6. D’Mello, J. P. F., Porter, J. K., MacDonald, A. M. C. and Placinta, C. M. 1997. Fusariunz mycotoxins. Ch. 20, pp. 287-301. In: Handbook of Plant and F~rngalTosicants. J. P. F. D’Mello (Ed.). CRC Press, Boca Raton, FL. 7. Abramson, D. 1997. Toxicantsof the genus Penicillium. Ch. 21. pp. 303-317. In: Handbook of Plant arzd Fungal Toxicants. J. P. F. D’Mello (Ed.). CRC Press, Boca Raton, FL. 8. Panigrahi, S. 1997. Altenzaria toxins. Ch. 32, pp. 319-337. In: Handbook of Plant am1 Fungal Tosicanrs. J. P. F. D’Mello (Ed.). CRC Press, Boca Raton, FL. 9. Visconti, A. and Sibilia. A. 1994. Alternaria toxins. Ch. 7. pp. 315-336. In: Mycotoxim in Grairz:Compozrnds Other Than Aflatoxin. J. D. Miller andH. L. Trenholm (Eds.). Egan Press, St. Paul, MN.

674

Porter

and Rotter, R. G. 1994. Toxicology of mycotoxins. Ch. 9, 10. Prelusky. D. B., Rotter, B. A. pp. 359-403. In: Mycotoxins in Grain: Corrtpounds Other Than AJlatoxin. J. D. Miller and H. L. Trenholm (Eds.). Egan Press, St. Paul, MN. 11. Kuipe-Goodman, T. 1994. Preventionof human mycotoxicoses through risk assessmentand risk management. Ch. 12. pp. 439-469. In: Mycotoxins irt Grain: Cornpolrrzds Other. Than AJlatoxin. J. D. Miller and H. L. Trenholm (Eds.). Egan Press, St. Paul, MN. 12. Beardall, J. M. and Miller, J. D. 1994. Diseases in humans with mycotoxins as possible causes. Ch. 14, pp. 487-539. In: Mycotoxins in Grain: Compounds Other Than Ajatoxin. J. D. Miller and H. L. Trenholm (Eds.). Egan Press, St. Paul, MN. 13. Bove, F. J. 1970. The Stoiy of Ergot. S. Karger, New York. 14. Berde, B. and H. 0. Schild. 1978. Ergot Alkaloids and Related Contpounds. Handbook ESP. Pharmacology, Vol. 49. Springer-Verlag, New York. 15. Bandyopadhyay, R., Frederickson, D. E., McLaren, N. W. and Ryley, M. J. 1998. Ergot: a new disease threat to sorghum in the Americas and Austrailia. Plant Dis. 82356-367. C. Hancock, J. G. 1975. Biological control of ergot by Fusarzl16. Mower, R. L., Snyder, W. and ium. Phytopathology 65:5-10. 17. Bacon, C. W., Porter, J. K. and Norred, W. P. 1995. Toxic interaction of fumonisin B, and fusaric acid as measured by injection into fertile chicken eggs. Mycopatlrology. 129:29-35. 18. Bacon, C. W.. Porter, J. K., Norred. W. P. and Leslie, J. F. 1996. Production of fusaric acid by Fusarium species. Appl. Environ. Microbiol. 624039-4043. 19. Porter, J. K., Wray, E. W., Rimando. A. M.. Stancel, P. C., Bacon, C. W. and Voss, K. A. 1996. Lactational passage of fusaric acid from the feed of nursing dams to the neonate rat and effects on pineal neurochemistry in the F1 and F2 generations at weaning, J. Toxicol. Enriron. Health 50275-284. 20. Porter, J. K., Bacon, C. W., Wray, E. M. and Hagler, W. M. Jr. 1995. Fusaric acidFusariunl in rnonilifontte cultures, corn, and feeds toxic to livestockand the neurochemical effects in the brain and pineal glands of rats. Nut. Tosirts 3:91-100. 21. Porter, J. K., Bacon, C. W.. Kuldau, G., Wray, E. M. and Meredith, F. I. 1998. Alkaloids and other mycotoxins associated with ergot damaged sorghum. Proceedings National Conference on Sorgltum Ergot, Corpus Christi, TX, June 25-26, 1998. (c~http://MlwlMl.ars-griiz.Qov/ i~lav/coilfernc/pol?er.l2tnz). and biochemical interactionsof the fungal metabolites fusaric 22. Dowd, P. F. 1988. Toxicologic acid and kojic acid with xenobiotics in Heliotlu's zea and Spodopterct fiwgiperda (J. Smith). Pesticide Biochem Physiol. 32: 123-134. 23. Taylor, L. T. 1996.Supercritical Fluid Extraction: Techniques in Analytical Chernistry. Wiley, New York. 24. Byrne, L. T. 1993. Nuclear magnetic resonance spectroscopy: strategies for structural determination. Ch. 4, p. 75. In: Bioacti]*eNatural Products: Detection, Isolation, and Structurul Determination. S. M. Colegate and R. J. Molyneux (Eds.). CRC Press, Boca Raton, FL. 25. Wong, R. Y. and Gaffield. 1993. Determinationof three-dimensional structure and configuration of bioactive natural compoundsby X-ray crystallography. Ch.6. p. 125. In: Bioactise Natural Products: Detection, Isolation, and Structural Determination. S. M. Colegate and R. J. Molyneux (Eds.). CRC Press, Boca Raton. FL. 26. Gaffield, W. 1993. Determination of the absolute configuration of bioactive natural cotnpounds, utilizing exciton chirality circular dichroism. 7., Ch.147. In: Bioactive Natural Products: Detection, Isolation, and Structural Determination. s. M. Colegate and R. J. Molyneux (Eds.). CRC Press, Inc., Boca Raton, FL. 27. Cole, R. J. and Cox, R. H. 1981. Handbook of Toxic Fungal Metabolites. Academic Press, New York. 28. Savard, M. E. and Blackwell, B. A. 1994. Spectral characteristics of secondary metabolites from Fusarium fungi. Ch. 4, pp. 59-257. In: Mycoto.yins in Grain: Compo~~nds Other Than AJlatoxin. J. D. Miller and H. L. Trenholrn (Eds.). Egan Press. St. Paul. MN.

Analytical Methodology for Mycotoxins

675

29. Turner, W. B. and Aldridge, D. C. 1983. Fungal Metabolites. Academic Press, New York. 30. Hofmann. A. 1964. Die Mutterkornalkaloide. Enke Verlag, Stuttgart. 31. Floss, H. G. 1976. Biosynthesis of ergot akaloids and related compounds. Tetrahedron 32: 873-912. 32. Porter, J. K. 1994. Chemical constituents of grass endophytes. Ch. 8, pp. 103-123. In: Biotechnology of Endophytic Fungi of Grasses. C. W. Bacon and J. F. White, Jr. (Eds.). CRC Press, Boca Raton, FL. 33. Porter, J. K. 1997. Endophyte alkaloids. Ch. 4, pp. 51-62. In: Handbook of Plant and Fungal Toxicants. J. P. F. D'Mello (Ed.). CRC Press, Boca Raton, FL. 35. Thompson, F. N. and Porter, J. K. 1991. Tall fescue toxicoxes in cattle: could there be a public health problem. Vef. Hum. Toxicol. 3251-57. 36. Stuedemann, J. A. and Hoveland, C. S. 1988. Fescue toxicity: history and impact on animal agriculture. J. Prod. Agric. 1:39-44. 37. Hoveland, C. S. 1993. Importanceand economic significance of Acrenzonirrn? endophytes to performance of animals and grass plants. In: Acremonium/Grass Interactions. R. E. Joost and S. S. Quisenberry (Eds.). Elsevier Scientific Publishers, Amsterdam, Netherlands. Agriculture, Ecosystems and Environment 44:3- 12. and Kerley, M. 1995. The effects of 38. Paterson, J., Forcherio, C., Larson, B., Samford, M. fescue toxicosis in beef cattle productivity. J. Anirn. Sci. 73:889-898. 39. Cheeke, P. R. 1995. Endogenous toxins and mycotoxins in forage grasses and their effects on livestock. J. Anim. Sci. 73:909-918. 40. Cross, D. L., Redmond, L. M. and Strickland, J. R. 1995. Equine fescue toxicosis: signsand solutions. J. Anim. Sci. 73:899-908. 41. Robbins, J. D., Porter, J. K. and Bacon, C. W. 1986. Occurrence and clinical manifestation of ergot and fescue toxicoses.In: Diagnosis ofMycotoxicoses. Ch. 6, pp 61-74. J.L. Richards and J. R. Thurston (Eds.). Martinus Nijhoff Publishers, Dordrecht, Netherlands. 42. Bacon, C. W., Lyons, P. C., Porter, J. K. and Robbins, J. D. 1986. Ergot toxicities from endophyte-infected grasses: a review. Agron. J. 78: 106-116. 43. Prestidge, R. A. 1993. Causes and control of perennial ryegrass staggers in New Zealand. In: Acremoniunz/Grass Interactions. R. E. Joost and S. S. Quisenberry (Eds.). Elsevier Scientific Publishers, Amsterdam, Netherlands. Agriculture, Ecosystems and Environment 44:283300. 44. Cunningham, P.J., Foot, J. Z. and Reed, K. F. M. 1993. Perennial ryegrass(Loliumperenne) endophyte (Acrentoniunz lolii) relationships:theAustralianexperience. In: Acrentonium/ Grass Interactions. R. E. Joost and S. S. Quisenberry (Eds.). Elsevier Scientific Publishers, Amsterdam, Netherlands.Agriculture, Ecosystents and Environment 44: 157- 168. In: Handbook of Naturally Occurring Food Toxicants. 45. Yates, S. G. Tall fescue toxins. 1983. pp. 249-273. M. Recheigl (Ed.). CRC Press, Boca Raton, FL. and Hanlin, R. T. 1996. Molecular phylogeny of Acrem46. Glenn, A.E., Bacon, C. W., Price, R. onium and its taxonomic implications. Mycologia 88:369-383. 47. Based on DNA sequence analyses,Acremonium coenophialu?n (Morgan-Jones & Gams), A. typhinunt (Morgan-Jones & W. Gams), A. lolii (Latch, Christensen& Samuels), A. chisosunt (J. F. White & Morgan-Jones), A. starrii (J. F. White & Morgan-Jones). and A. unicincrtum (W. Gams, Petrini & D. Schmidt) have been reclassified as: Neotyphodiurn coenopltialum-, N. typhinum-, N. lolii-, N. chisosum-, N.stcrrrii-, and N. unicinatum-Glenn, Bacon, & Hanlin comb. nov.. respectively. A dual system of nomenclature differentiates Neotyphodium and the sexually reproducing species classified in Epichloe. 48. Neotyphodiunt-Grass Interactions. C. W. Bacon and N. S. Hill (Eds.). 1997. Proceedingsof the Third International Symposium on AcremoniumlGrass Interactions, May 28-3 1, 1997, Athens, GA. Plenum Press, New York. 49. Porter, J. K. and Thompson, F. N. Jr. 1992. Effects of fescue toxicosis on reproduction in livestock. J. Anirn. Sci. 70:1594-1603.

676

Porter

and their metabolitesas extrinsic factors 50. Bacon, C. W. 1994. Fungal endophytes, other fungi, of grass quality. Ch. 8, pp. 318-366. In: Forage Qualio. Evnl~mtio~t a d Utilization. G. C. Fahey Jr. (Ed.). American Society of Agronomy, Madison, WI. of Epiclzloe typhirza and Acremo51. Schardle, C. L.and Siegel, M. R. 1993. Molecular genetics rzium coelzophialurn. In: AcrenzoniludGrass Interactions, R. E. Joostand S. S. Quisenberry (Eds.). Elsevier Scientific Publishers, Amsterdam, Netherlands. Agriculture, Ecosystems and Enliroilment 44:169-185. 53. Perellino, N. C . , Malyszko, J., Ballabio, M., Gioia, B.and Minghetti, A. 1992. Direct biosynthesis of unnatural ergot alkaloids. J. Not. Prod. 55:424-427. 54. Perellino, N. C.. Malyszko. J., Ballabio, M., Gioia, B. and Minghetti.A. 1993. Identification of ergobine, a new natural peptide ergot alkaloid. J. Nat. Prod. 56:489-493. 55. Flieger, M., Wurst. M., Stuchlik, J. and Rehacek, Z. 1981. Isolation and separation of new natural lactam alkaloids of ergot by high performance liquid chromatography. J. Chronzatogr. 207:139-144. 56. Porter, J. K., Bacon, C. W., Meredith, F. I., Wray, E. M. and Wilson, J. P. 1998. G U M S analysis of the TMS-clavine alkaloids in Claviceps africana-infected sorghum from Georgia. 112'hAOAC International Annual Meeting and Exposition, Montreal, Quebec, Canada, Sept. 13-17, 1998. p. 65, A-201 (abstract). 57. Powell, R. G., Plattner. R. D., Yates, S. G., Clay, K. and Leuchtmann, A. 1990. Ergobalansine, a new ergot-type peptide alkaloid isolated from Cenchrus ecfzinatus (sandbur grass) infected with Balansia obtecta, and produced in liquid cultures of B. obtecta and Balansia cyperi. J. Nut. Prod. 53:1272-1279. 58. Yates, S. G., Plattner, R. D. and Garner, G. B. 1985. Detection of ergopeptine alkaloids in endophyte infected, toxic K-3 1 tall fescue by mass spectrometry/mass spectrometry.J. Agric. Food Chem. 33:719-722. 59. Yates, S. G. and R. G. Powell. 1988. Analysisof ergopeptine alkaloidsin endophyte-infected tall fescue. J. Agric. Food Chem. 36:337-340. 60. Lyons, P. C., Plattner, R. D. and Bacon, C. W., 1986. Occurrence of peptide and clavine ergot alkaloids in tall fescue grass. Science 232487-489. 61. Petroski, R. J. and Powell, R. J. 1991. Preparative separation of complex alkaloid mixture by high-speed countercurrent chromatography.In: Naturally Occurring Pest Bioregulmors. Paul A. Hedin (Ed.) Am. Chem. SOC. Symposium Series #449. p 326. 62. Plattner, R. D., Yates, S. G. and Porter, J. K. 1983. Quadrupole mass spectrometry/mass spectrometry of ergot cyclol alkaloids. J. Agric. Food Client. 31:785-789. 63. Porter, J. K., Bacon, C. W. and Robbins.J. D. 1979. Ergosine, ergosinine,and chanoclavine I from Epichloe typlzina. J. Agric. Food Chem. 27:595-598. 64. Porter, J. K., Bacon, C. W., Robbins.J. D. and Betowski, D. 1981. Ergot alkaloid identification in clavicipitaceae systemic fungi of pasture grasses. J. Agric. Food Clwm. 29:653657. 65. Rottinghaus, G. E.. Garner, G. B., Cornell. C. N. and Ellis, J. L. 1991. An HPLC method for quantitating ergovaline in endophyte-infected tall fescue: seasonal variationof ergovaline levels in stems with leaf sheaths,leaf blades and seed heads.J. Agric. Food Clzem. 39: 112115. 66. Shelby, R. A. and Fleiger. M. 1997. Analysisof ergot alkaloids in plantsand seeds of endophyte-infected tall fescue by gradient HPLC. Ch.50, pp. 271-273. In: Neot?;ol?odiroiz/Grass I~lterrrctions.C. W. Bacon and N. S. Hill (Eds.). Proceedingsof the Third International Symposium on AcrernoniunzlGrass Interactions, May 28-3 1, 1997, Athens, GA. Plenum Press, New York. 67. Groger. D., Erge, D. and Floss, H. G. 1968. Uber die herfkunft der seitkette D-lySergSaUreim methlcarbinolamide. Zeitschr. Nccturforsclt. B. 23B: 177-180. 68. Scott, P. M., Lombaert, G. A., Pellaers, P., Bacler, S. and Lappi, J. 1992. Ergot alkaloids in grain foods sold in Canada. J. AOAC Int. 75:773.

Analytical Methodology for Mycotoxins

677

69. Gamer, G. B., Rottinghaus, G. E., Cornell, C. N. and Testereci, H. 1993. Chemistry of compounds associated with endophyte/grass interaction: ergovaline- and ergopeptine-related alkaloids. Agric. Ecosyst. Erniron. 44:65-80. 70. Porter, J. K.. Bacon, C. W., Plattner, R. D. and Arrendale, R. F. 1987. Ergot peptide alkaloid spectra of Claviceps-infected tall fescue, wheat, and barley. J. Agric Food Clzem. 35:359-361. 71. Porter, J. K.. Bacon, C. W. and Robbins, J. D. 1974. Major alkalodis of a Claviceps isolated from toxic bermuda grass. J. Agric. Food Cl~ent.228384341. 72. Testereci, H., Garner, G. B., Rottinghaus, G. E., Cornel, C. N. and Andersen, M. P. A. 1990. Method for large scale isolation of ergovaline from endophyte-infected tall fescue seed (Festuca arzrndinacea). In: S.S. Quisenbeny and R. E. Joost (Eds.). Proc. Int. Symp.on AcremoniumlGrass Interactions, New Orleans, LA, Nov. 3, 1990. p 100. Louisiana Agricultural Experiment Station, Baton Rouge. 73. Moubarak, A. S., Piper, E. L., West, C. P. and Johnson, Z. B. 1993. Interaction of purified ergovaline from endophyte-infected tall fescue with synaptosomal ATPase enzyme system. J. Agl-ic. Food Cllenz. 41:407-409. 74. Belesky, D. P., Stuedemann, J. A., Plattner, R. D. and Wilkinson, S. R. 1988. Ergopeptine alkaloids in grazed tall fescue. Agron. J. 80:209-212. 75. Lyons, P. C., Evans, J. J. and Bacon, C. W. 1990. Effects of fungal endophyte Acrenzonizrm coenophialzrm on nitrogen accumulation and metabolism in tall fescue. Plant. Pltpiol. 92: 726-732. 76. Archavaleta, M., Bacon, C. W., Plattner, R. D., Hoveland, C. S. and Radcliffe, D. E. 1992. Accumulation of ergopeptide alkaloids in symbiotic tall fescue grown under deficits of soil water and nitrogen fertilizer. Appl. Emiron. Microbiol. 58:857-861. 77. Hill, N. S., Parrott, W. A. and Pope, D. D. 1991. Ergopeptine alkaloid production by endophytes in a common tall fescue genotype. Crop. Sci. 3 1:1545-1547. 78. Hill, N. S., Rottinghaus, G. E., Agee, C. S. and Schultz, L. M.. 1993. Simplified sample preparation for HPLC analysis of ergovaline in tall fescue. Crop Sci. 33:331-333. 79. Zhang, Q., Spiers, D. E., Rottinghaus, G. E. and Garner, G. B. 1994. Thermoregulatory effects of ergovaline isolated from endophyte infected tall fescue seed on rats. J. Agric. Food Chent. 42:954-958. 80. Scott, P. M. and Lawrence, G. A. 1980. Analysis of ergot alkaloids in flour. J. Agric. Food Cltem. 28:1258. 81. Christensen, M. J., Lane, G. A., Simpson, W. R. and Tapper, B. A. 1997. Leaf blade colonization by two Neotyphodium endophytes, and ergovaline distribution within leaves of tall fescue and meadow fescue. Ch. 23, pp. 149-151. In: C. W. Bacon and N. S. Hill (Eds.). Neot?lplzodiuliz/GI-nss Interactions. Plenum Press, New York. 82. Rottinghaus, G. E., Schultz. L. M., Ross, P. F. and Hill, N. S. 1993. An HPLC method for the detection of ergot in ground and pelleted feeds. J. Vet. Diagn. Intvst. 5:242. 83. Stahl, E. 1969. Tlzirt Layer Clzrornatograplty,A Laboratory Handbook, No. 73. pp. 127, 869. Springer-Verlag, New York. 84. Sprince, H. 1960. A modified Ehrlich benzaldehyde reagent for detection of indoles on paper chromatograms. J. Cltrontatogr. 3:97-98. 85. Michelon, L. E. and Kelleher, W. J. 1963. The spectrophotometric determination of ergot alkaloids. A modified procedure employing paradimethylaminobenzaldehyde. Lloydia 26: 192-201. 86. Porter, J. K. and Betowski, D. 1981. Chemical ionization mass spectrometry of the ergot cyclol alkaloids. J. Agric. Food Chent. 29:650-653. 87. Bianchi, M. L., Perellino, N. C., Gioia, B. and Minghetti, A. 1982. Production by Clmiceps purpurea of two new peptide ergot alkaloids belonging to a new series containing alphaaminobutyric acid. J. Nut. Prod. 45: 191-196. 88. Rowan, D. D. and Shaw, G. J. 1987. Detection of ergopeptine alkaloids in endophyte-infected perennial ryegrass by tandem mass spectrometry. NZ Vet. J . 35:197-198.

678

Porter

J. 1992.Theloline groupofpyrrolizidinealkaloids. In: 89. Powell,R.G. andPetroski,R. S. W. Pelletier (Ed.). The Alkaloids: Chemicaland Biological Persl>ectilyes. Vol. 8. pp. 320338. Springer-Verlag. New York. 90. Yates, S. G., Petroski, R. J. and Powell, R. G. 1990. Analysis ofloline alkaloids in endophyteinfected fescue by capillary gas chromatography. J. Agric. Food Clzem. 38:182-185. 91. Siegel, M. R., Latch, G. C. M., Bush, L. P., Fannin, F. F., Rowan, D. D., Tapper, B. A., Bacon, C.W. and Johnson. M. C.1900. Fungal endophyte-infected grasses: alkaloid accumulation and aphid response. J. Cltem. Ecol. 123301-3315. 92. Petroski, R. J., Dornbos D.L., Jr. and Powell, R. G.1990. Germination and growth inhibition of annual ryegrass (Loliunt mdtiJlorunz L.) and alfalfa (Medicngo sativa L.) by loline alkaloids and synthetic N-acetylloline derivatives. J. Agric. Food Clzerrz. 38:1716-1718. 93. Bush, L. P., Fannin, F. F., Siegel. M. R., Dahlman,D. L. and Burton, H. R. 1993. Chemistry, occurrence and biological effects of saturated pyrrolizidine alkaloids associated with endophyte-grass interactions. Agric. Ecosyst. Emiron. 44:8 1- 102. 94. Tepaske. M. R., Powell, R. G.and Petroski, R. J. 1993. Quantitative analysis of bovine urine and blood plasma for loline alkaloids. J. Agric. Food Chertz. 4 1 2 3 1-234. 95. Eichenseer, H.,Dahlrnan. D. D. and Bush, L. P. 1991.Influenceof endophyte infection, plant age and harvest interval on RhopaZosipl?uvz pndi survival and its relation to quantity of N-formyl and N-acetyl loline in tall fescue. Entomol. Esy. Appl. 6029-38. 96. Belesky, D. P.. Robbins, J. D., Stuedemann, J. A., Wilkinson, S. R. and Devine, 0. J. 1987. Fungal endophyte infection-loline derivativealkaloidconcentrationofgrazed tall fescue. Agron. J. 79:217-220. 97. Gallagher. R. T., Hawkes, A. D. and Stewart, J. M.. 1985. Rapid determination of the neurotoxin lolitrem B in perennial ryegrass by high-performance liquid chromatography with fluorescence detection. J. Cltromatogr. 321:217-226. 98. Weedon, C. M. and Mantle, P.M. 1987. Paxilline biosynthesisby Acrentorzizut loliae; a step toward defining the origin of lolitrem neurotoxins. Plzytochenzistv 26:969-971. 99. Miles, C. O., Wilkins, A. L.. Gallagher, R. T., Hawkes, A. D.,. Munday, S. C and Towers, N. R. 1992. Synthesis and tremorgenicityofpaxitrols and lolitriol: possiblebiosynthetic precursors of lolitrem B. J. Agric. Food Chem. 40234-238. 100. Fletcher, L. R., Garthwaite. I. and Towers. N. R. 1993. Ryegrass staggers in the absence of lolitrern B. In: D. E. Hume, G. C. M. Latch, and H. S. Easton (Eds.) Proc. 2nd Int. Symp. on Acrerrzortiur~t/Grass Interactions.Massey University, Feb. 4-6, 1993. p 119. AgResearch, Palmerston North, New Zealand. 101. Penn, J.. Garthwaite, I., Christensen, M. J., Johnson. C. M. and Towers, N. R. 1993. The importance of paxilline in screening for potentially tremorgenic Acrernortiun isolates. In: D. E. Hume. G. C. M. Latch, and H. S. Easton (Ed.) Proc. 2nd Int. Symp. on Acrentoniud Grass Interactions. Massey University, Feb. 4-6, 1993. p 88. AgResearch, Palmerston North, New Zealand. 102. Miles, C. O., Munday, S. C., Wilkins. A. L., Ede, R. M., Hawkes, A. D., Embling. P. P. and Towers, N. R. 1993. Large scale isolation of lolitrem B, structure determination of some minor lolitrems, and tremorgenic activities of lolitrem B and paxilline in sheep. In: D. E. Hume, G. C. M. Latch and H. S. Easton (Eds.). Proc. 2nd Int. Synlp. on Acremonium/Grass Interactions, Massey University, Feb. 4-6, 1993. p 85. AgResearch, Palmerston North,New Zealand. 103. Tapper, B. A., Rowan, D. D. and Latch, G. C. M. 1989. Detection and measurement of the alkaloid peramine in endophyte-infected grasses. J. Chromatogr. 463: 133-138. 104. Rowan, D. D. and Tapper, B. A. 1989. An efficient method for the isolation of peramine, an insect feeding deterrent produced by the fungus Acrmtonium lolii. J. Not. Prod. 52:193195. 105. Rowan, D. D. 1993. Lolitrems, peramine and paxilline: mycotoxins of ryegrass/endophyte interaction. Agric. Ecosyst. Erniron. 44: 103-122.

Analytical Methodology for Mycotoxins

679

106. Rowan, D. D.. Hunt, M. B. and Gaynor, D. L. 1986. Peramine, a novel insect feeding deterrent from lyegrass infectedwiththe endophyte Acrenroniunr loliae. J Chem. SOC.Clzem. COt?1ti1lc?z.935-936. 107. Fannin, F. F., Bush, L. P., Siegel, M. R. and Rowan, D. D. 1990. Analysis of peramine in fungalendophyte-infectedgrassesbyreversed-phasethin-layer chromatography. J. Cl11-0matogr. 503:288-292. 108. Kellerman. T. S., Marasas, W. F. O., Thiel, P. G., Gelderblom. W. C. A., Cawood, M. and Coetzer. J. A. W. 1990. Leukoencephalomalacia in two horses induced by oral dosing of fumonisin B1. Onderstepoort J. Vet. Res. 57:269-275. 109. Bezuidenhout, S. C., Gelderblom, W. C. A., Gorst-Allman, C. P., Horak. R. M., Marasas, W. F. 0..Spiteller, G.and Vleggaar, R. 1988. Structure elucidation of the fumonisins, mycotoxins from Fmariunz ~~orzilifornle. J. Chenr. SOC.Chem. Conrrmn. 743-745. 110. Norred, W. P. and Voss, K. A., 1994. Toxicity and role of fumonisins in animal diseases and human esophageal cancer. J. Food Protect. 57:522-527. 111. Gelderblom, W. C. A., Jaskiewicz, K., Marasas, W. F. O., Thiel, P. G., Horak,R. M., Vleggaar, R. and Kriek. N. P. J. 1988. Fumonisins-novel mycotoxins with cancer-promoting activity produced by Fusarium monilifornre. Appl. Errviron. Microbiol. 54: 1806- 18 11. 112. Youshizawa, T., Yamashita, A. and Luo, Y. 1994. Fumonisin occurrence in corn from highand low-risk areas for human esophageal cancer in China. Appl. Environ. Microbiol. 60: 1626. 113. Franceschi, S., Bidoli, E., Baron, A. E. and La Vecchia, C. 1990. Maize and risk of cancers of the oral cavity, pharynx, and esophagus in northern Italy. J. Cancer Inst. 821407. 114. Gelderblom, W. C. A., Kriek, N. P. J., Marasas, W. F. 0. and Thiel. P. G. 1991. Toxicity and carcinogenicity of theFusariwn moniliforme metabolite, fumonisin B 1, rats. in Carcinogenesis 12:1247-1251. 115. Marasas, W. F. O., Jaskiewicz, K., Venter, F. S. and van Schalkwyk, D. J. 1988. Fusarium monilifonne contamination of maize in oesophageal cancer areas in Transkei. S. A@. Med. J. 74:llO-114. 116. Thiel, P. G.,Marasas, W. F. 0.. Sydenham, E. W., Shephard,G. S. and Gelderblom, fumonisins in corn for W. C. A. 1992. The implicationsofnaturally-occurringlevelsof human and animal health. Mycopntlzologia 117:3-9. 117. Riley, R. T.. Voss, K. A.. Yoo, H.. Gelderblom. W. C. A. and Merrill,A. H. 1994. Mechanism of fumonisin toxicity and carcinogenesis. J. Food Protect. 57:638. 118. Riley, R. T., Norred, W. P. and Bacon, C. W. 1993. Fungal toxins in foods: recent concerns. Annu. Rev. Nzrtr. 13: 167-1 89. 119. Bacon, C. W. and Nelson, P. E. 1994. Fumonisin production in corn by toxigenic strains of Fmn-iunr lnorriliforme and Fusarimr proliferatum. J. Food Protect. 5 7 5 14-521. 120. Sydenham, E. W., Shephard, G. S., Theil, P. G., Marassas. F. O., Rheeder, J. P., Sanhueza, C. E. P., Gonzalez, H. L. and Resnik, S. L. 1993. Fumonisins in Argentinian field-trial corn. J. Agric. Food. Chem. 41:89 1. 121. Plattner, R. D., Norred, W. P., Bacon, C. W., Voss, K. A., Peterson, R., Shackelford, D. D. and Weisleder, D. 1990. A method of detection of fumonisins in corn samples associated with field cases of leukoencephalomalacia. Mycopatlzologia 82:698-702. 122. Drysdale, R. B. 1984. The production and significance in phytopathology of toxins produced by species of Fusar-iuw. In: The Applied Mycology of Fusarium, p. 95. M. 0. Moss and J. E. Smith (Eds.). Cambridge University Press, New York. Ch. 1, pp. 3- 18. 123. ApSimons, J. 1994. The biosynthetic diversity of secondary metabolites. In: Mycotoxins in Grain: Cortzpounds Other Than Ajlatoxin. J. D. Miller and H. L. Trenholm (Eds.). Egan Press, St. Paul, MN. 124. Bacon, C. W.,Bennett,R.M., Hinton, D. M. and Voss, K. A.1992. Scanning electron microscopy of Fusarinnt moniliforrire within asymptomatic corn kernels and kernels associated with equine leukoencephalomalacia. Plalrt Dis. 76: 144-148.

680

Porter

125. Doko. M. B., Canet, C., Brown, N., Sydenham, E. W., Mpuchane, S. and Siame, B. A. 1996. Natural occurrence of fumonisins and zearalenone in cerals and cereal-based foods from Eastern and Southern Africa. J. Agric. Food Clzem. 44:3240-3243. 126. Gelderblom. W. C. A., Marasas, W. F. O., Thiel, P.G.. Veggaar, R. and Cawood, M. E. 1992. Fumonisins, chemical characterization and biological effect. Mycopathologia 117:11-1 4. 127. Meredith, F., Bacon C., Plattner, R. and Norred, W. 1996. Preparative LC isolation and purification of fumonisin B1 from rice cultures. J. Agric. Food Chem. 44:195-198. 128. Meredith, F., Bacon C.. Norred, W. and Plattner, R. 1997. Purification of fumonisin B2 isolated from rice culture. J. Agric. Food Chem. 45:3143-3147. 129. Holcomb, M., Thompson, H. C.. Jr. and Hankins, L. J. 1993. Analysis of fumonisisn B1 in rodent chow by gradient elution HPLC using precolumn derivatization with FMOC and fluorescence detection. J. Agric. Food Chem. 41:764-767. 130. Sydenham, E. W., Shepard, G. S., Thiel, P. G., Bird, C. and Miller. B. M. 1996. Determination of fumonisins in corn: evaluation of competitive inmunoassay and HPLC techniques. J. Agi-ic. Food Chem. 44: 159-164. 131. Sydenham, E. W.,Thiel, P. G., Shepard, G. S.. Koch, K. R. and Hutton, T. 1995. Preparation and isolation of the partially hydrolyzed moiety of fumonisin B 1. J. Agric. Food Chenr. 43: 2400-2405. 132. Sydenham E. W., Shepard, G. S. and Thiel, P. G. 1992. Liquid chromatographic determinations of fumonisins B,, B2, and B? in foods and feeds. J. Assoc. Off Anal. Chenz. Int. 75: 313-318. 133. Scott, P. M. and Lawrence, G. A. 1992. Liquid chromatographic determination of fumonisins with 4-fluoro-7-nitrobenzofurazan. J. Assoc. Off Anal. Clzem. 75:829-834. 134. Shepard, G. S.. Sydenham, E. W., Thiel, P. G. and Gelderblorn, W. C. A. 1990. Quantitative determination of fumonisins B1 and B2 by high-performance liquid chromatography with fluorescence detection. J. Liq. Chromatogr-. 13:2077-2087. 135. Stack M. E. and Eppley, R. M. 1992. Liquid chromatographic determination of fumonisins B1 and B2 in corn and corn products. J. Assoc. OffAnd. Chem. 75:834-837. 136. Cawood, M. E., Gelderblom, W. C. A., Vleggaar, R., Behrend, Y., Thiel, P. G. and Marasas, W. F. 0. 1991. Isolation of the fumonisin mycotoxins: a quantitative approach. J. Agric. Food C I I ~ I39: ? ~1958-1962. . 137. Vesonder, R., Patterson, R., Plattner, R. and Weisleder, D. 1990. Fumonisin B1: islolation from corn culture, and purification by high performance liquid chromatography. Myoto.xin Res. 85-88. 138. Meredith, F., Bacon C., Nolred,W. and Plattner, R. 1996. Isolation and purification of fumonisin B1 and B2 from rice culture. Ch. 10, p. 113-122. In: Fzlmonisins irz Food. L. Jackson (Ed.). Plenum Press, New York. 139. Rottinghaus, G. E., Coatney, C. E. and Minor, H. C. 1992. A rapid, sensitive thin layer chromatography procedure for the detection of fumonisin B, and B?. J. Vet. Dirrgn. Invest. 4:326-329. 140. Musser, S. M., Eppley, R. M., Mazzola, E. P., Hadden, C. E., Shockcor, J. P., Crouch, R. C. and Martin, G. E. 1995. Identification of an N-acetylketo derivative of fumonisin B1 in corn cultures of Fusarium prolifemtunz. J. Not. Prod. 58:1392-1397. 141. Meredith, F. I. 1999. Isolation and characterization of fumonisins. Vol. 311, pp. 361-373. In: Methods in Enzymology: Sphirlgolipid Metabolism and Cell Signaling. Y. A. Hannun and A. H. Merrill. Jr. (Eds.). Academic Press, Orlando, FL. 142. Haschek, W. M.and Haliburton, J. C. 1986.Fusarium nloniltforrneand zearalenone toxicoses in domestic animals: a review. Ch. 20, pp. 213-235. In: J. L Richards and J. R. Thurston, J. R. (Eds.). Diagnosis of Mycoto.vicoses. Martinus Nijhoff Publishers. Dordrecht, Netherlands. 143. Bennett, G. A., Shotwell, 0. L. and Kwolek, W. F. 1985. Liquid determination of a-zearalenol and zearalenone in corn: collaborative study. J. Assoc. Of Anal. Chem. 68:958-961.

Analytical Methodology for Mycotoxins

681

144. Kuiper-Goodman, T.. Scott, P. M.and Watanabe, H. 1987. Risk assessmentof the mycotoxin zearalenone. Regul. Toxicol. Pharnzacol. 7:253-306. 145. Ryu, J-C., Yang, J-S., Song, Y-S., Kwon, 0-S., Park, J. and Chang, I-M. 1996. Survey of natural occurrence of trichothecene mycotoxinsand zearalenone in Korean cereals harvested in 1992 using gas chromatography/mass spectrometry. Food Addit. Contam. 13:333-341. 146. Seo, J-A., Kim, J-C., Lee, D-H.and Lee, Y-W. 1996. Variations in 8-ketotrichothecenesand zearalenone production by Fusarium graminearum isolates from corn and barley in Korea. Mycopathologia 134:3 1-37. 147. Tanaka, T., Hasegawa. A., Matsuki, Y., Ishii, K., Ueno, Y. 1985. Improved methodology for the simultaneous detectionof trichothecene mycotoxins deoxynivalenoland nivalenol in cereals. Food Addit. Contam. 2125-137. 148. Rava, E. 1996. Mycotoxins in maize productsof the 1994/ 1995 marketing season. Mycotoxin Res. 1225-30. 149. Wang, D-S., Liang, Y-X., Chau, N. T., Dien, L. D., Tanaka, T. and Ueno, Y. 1995. Natural co-occurrence of Fusarium toxins and aflatoxin B1 in corn for feed in North Vietnam.Nat. Toxins 3:445-449. 150. Grabarkiewicz-Szczesna, J., Foremska, E. and Golinski. P. 1996. Distribution of trichothecene mycotoxinsin maize ears infected with F. gramirzearum and F. cookwellense. Mycotoxin Res. 12:45-50. 151. Rotter, B. A., Prelusky, D. B. and Pestka, J. J. 1996. Toxicology of deoxinivalenol (vomitoxin). J. Toxicol. Ernliron. Health 48: 1-34. 152. Rotter. B. A., Prelusky, D. B. and Thompson, B. K. 1996. The role of tryptophan in DONinduced feed rejection. J. Emyiron. Sci. Health B31(6):1279-1288. 153. Prelusky, D. B. 1994. The effect of deoxynivalenol on serotonergic neurotransmitter levels in pig blood. J. Emiron. Sci. Health B29(6):1203-1218. 154. Thiel, P. G., Meyer, C. J. and Marasas, W. F. 0. 1982. Natural occurrence of moniliformin together with deoxynivalenoland zearalenone in Transkeian corn.J. Agric. Food Clzem. 30: 308-312. 155. Snijders. C. H. A. and Perkowski, J. 1990. Effects of head blight caused by Fusariunz culmorum on toxin content and weight of wheat kernels. Phytopathology 80:566-570. 156. Perkowski, J., Kiecana, I. and Chelkowski. J. 1995. Susceptibility of barley cultivars and lines to Fmarizsm infection and mycotoxin accumulation in kernels. J. Phytopathol. 143: 547-555. 157. Miller, J. D., Savard. M. E., Sibilia. A.,Rapior, S., Hocking, A. D. and Pitt, J. I. 1993. Production of fumonisns and fusarins by Fusarium modiforrm from Southeast Asia.Mycologia 85:385-381. 158. Savard. M. E. and Miller, J. D. 1992. Characterization of fusain F, a new fusarin from Fusarium monilifonne. J. Nat. Prod. 55:64-70. 159. Gupa, S., Krasnoff, S. B., Underwood, N. L., Renwick, J. A. A. and Roberts. D. W. 1991. Isolation of beauvericin as an insect toxin from Fusarium semitectunt and Fusarium nroniliforme var. subglz4tinans. Mycopathologia 1 15:185-1 89. 160. Ritieni, A., Fogliano, V., Randazzo, G., Scarallo, A., Logrieco, A., Moretti, A., Mannina, L. and Bottalico, A. 1995. Isolation and characterization of fusaproliferin, anew toxic metabolite from Fusariwt proliferaturn. Nat. Toxins 3: 17. 161. Xie, W., Mirocha, C. J., Wen, Y., Cheong, W-J. and Pawlosky, R. J. 1991. Isolation and structure elucidation of four fatty acid derivatives of the mycotoxin fusarochromanone produced by Fusarium episeti. J. Agric. Food Chenz. 39:1757-1761. 162. Xu, Y., Mirocha, C. J. and Zie, W. 1993. Analysisby liquid chromatography of fusarochromanone (TDP-I) added to corn and wheat. J. Assoc. 08 Anal. Chem. Int. 77: 1 179-1 183. 163. Scott, P. M., Abbas, H. K.. Mirocha, C. J., Lawrence, G. A. and Weber, D. 1987. Formation of moniliformin by Fusarium sporotrichioides and Fusariurtz culrnorum. Appl. Emiron. Microbiol. 53: 196-1 97.

682

Porter

164. Scott, P. M., Kanhere, S. R. and Tarter, E. J. 1986. Determinationof nivalenol and deoxynivalenol in cereals by electron-capture gaschromatography.J. Assoc. Qfl Anal. Cltem. 64:961963. 165. Sydenham, E. W. and Thiel, P. G. 1987. The simultaneous determination of diacetoxyscirpenol and T-2 toxin in fungal cultures and grain samples by capillary gas chromatography. Food Addit. Contant. 4:277-284. 166. Sydenham, E. W., Thiel, P. G. and Marasas, W. F. 0. 1988. Occurrence and chemical determination of zearalenone and alternariolmonomethyl ether in sorghum based mixed feeds associated with an outbreak of hyperestrogenism in swine. J. Agric. Food Chent. 36:621625. 167. Roberts, B. S., Glancy, E. M. and Patterson, D.S.P. 1981. Rapid and economical method for determination of aflatoxin and ochratoxin in animal feedstuffs. J. Assoc. 08 Anal. Chent. 64:961-963. 168. Trucksess, M. W., Stack, M.E., Nesheim, S., Page, S. W. and Albert, R. A. 199 1. Immunoaffinity column coupled with solution fluorometry or liquid chromatography postcolumn derivatization for the determination of aflatoxins in corn, peanutsand butter: collaborative study. J. Assoc. 08 Anal. Chem. 74:81-88. 169. Wang, D-S., Liang, Y-X, Chau, N. T., Dien, L. D., Tanaka, T. and Ueno, Y. 1995. Natural co-occurrence of Fusariun? toxins and aflatoxin B1 in corn for feed in North Vietnam. Nat. Toxins 3:445-449. 170. Frisvad. J. C. 1987. High performance liquid chromatographic determination of profiles of mycotoxins and other metabolites. J. Chrornatogr. 392:333-347.

17 Mycotoxin Analysis: Immunological Techniques Fun S. Chu Urlillersi@of Wiscorzsin-Madisorz, Madison, Wisconsin

684 I. Introduction 11. GeneralConsiderations684 A. Availability of antibody and markers 685 B. Adequate method for the separation of free and bound toxin 685 C.Understanding the specificity of the antibodies used in the assay 685 D. Understanding the effect of matrix and solvent system on the assay 686 111. Radioimmunoassay 687 A. B. C. D.

Principles 688 Preparation of radioactiveligands used in the RIA of mycotoxins688 Separation of Ab-mycotoxincomplexfromfreemycotoxins688 Application of RIA formycotoxins689

IV. Enzyme Itnmunassay A. B. C. D.

689

General considerations and assay configurations 689 Direct competitive ELISA (dc-ELISA) 690 Indirectcompetitive ELISA (idc-ELISA or double-antibodyELISA) Considerations of sampletreatment in the ELISA692

691

V. ImmunoscreeningMethods693 VI. Complementation of Immunoassays with Chemical Methods 694 A. Immunoaffinity chromatography 694 B. Combination of immunoassay with other chemical methods

695

VII. Other Newly DevelopedImmunochemicalMethods696 A. Anti-idiotype antibody-based immunoassays 696 B. Immunoassay for mycotoxin-producing fungi 697 C. Development of biosensors 697 VIII. Concluding Remarks 699 References 700

683

684

1.

Chu

INTRODUCTION

Mycotoxins are low-molecular-weight, secondary metabolites produced by naturally occurring fungi (1-8). Since the discovery of aflatoxins in the early 1960s (1, 6), developments in the last three decades have disclosed many new fungal poisons that are attracting attention because of their high toxicity and their association with foods and animal feeds (1-8). The presence of mycotoxins in foods and feeds is potentially hazardous to human and animal health. To decrease the risk of human exposure to mycotoxins, a rigorous program has been established for monitoring these toxins in foods. Governments in most countries have established limits for the levels of a number of mycotoxins that are permissible in foods and feeds and official methods of analysis for many mycotoxins have also been established (9, 10). However, because only trace amounts of the toxin are present in the sample, analysis of mycotoxins in foods is difficult. Research attempting to develop more sensitive, specific, and simple methods for mycotoxin detection has been done over the years. Rapid progress in the area of mycotoxin analysis has been made during the last few years (1 1- 16). The progress of such research can be seen from the numerous papers cited by the "General Referee on Mycotoxins," which appears in every year's January/ February issue of the J o w m l of the Association of OfJicial Analytical Chemists, Intermtioml (JAOAC Int.) (e.g., 17-20), and in several recent reviews and books including a separate chapter in this book. As has been discussed in another chapter, extensive sample cleanup treatment is needed for most chemical methods for mycotoxin analysis and they are very time consuming and need expensive instruments. Although several biological methods are available, most of these methods are nonspecific and relatively insensitive. To overcome the difficulties encountered with the chemical and biological methods, new immunochemical methods have been developed (21-37). Within the last few years, wide application of immunoassays for mycotoxins has been noted (22, 24-28, 36, 37). There has been a rapid increase in publications in this area. For example, in the 1991 and 1994 AOAC mycotoxin-associated referee reports, as many as 25-28% of the cited publications on mycotoxin analysis were immunoassay-related articles. More than 30% ofpublications on the analytical methods for mycotoxins now involve immunochemical techniques that either serve as a cleanup step or are used directly as a screening method or for quantitation (17-20). Many immunoassay kits for mycotoxin analysis are also commercially available (14, 22, 24, 36, 37). The application of mycotoxin immunoassay is not limited to foods and feeds: it has been used as a sensitive approach to monitoring mycotoxins in body fluids and tissues or organs of humans and animals that have been exposed to the mycotoxins (14, 24, 36). Thus, a new dimension of methodology for mycotoxin analysis as well as a new tool for diagnosis of mycotoxicoses in humans and animals has emerged since we first initiated a research project to develop an immunoassay for mycotoxins in the early 1970s (21-23, 29, 31). In this chapter, the general principles and recent applications of immunoassay for mycotoxins will be discussed. For a detailed discussion on immunoassays and earlier literature, several of the most recent reviews should be consulted (14, 23, 26, 30-36). For the specificity of antibodies against various mycotoxins, see Chu (14, 24, 26, 28).

II. GENERALCONSIDERATIONS

Immunochemical methods are based on the specific interaction between antibodies and the toxin. However, mycotoxins are low-molecular-weight compounds and they are not

Mycotoxin Analysis

685

immunogenic. Like most other natural products, mycotoxins must first be conjugated to a protein/polypeptide carrier before subsequent use in immunization for antibody production (14, 31, 23, 24, 26-28). Extensive studies have been done on the development of methods of conjugation of mycotoxins to a protein or polypeptide carrier and optimization of conditions for antibody production in rabbits and other animals (polyclonal antibodies). Newer innovative approaches for the coupling of mycotoxin to marcomolecule have been developed in recent years (28). With the advances in hybridoma technology, monoclonal antibodies against many mycotoxins were also made. Antibodies against almost all the important mycotoxins have been made available. Many types of immunoassays, including radioimmunoassay (RIA) and enzyme-linked immunosorbent assays (ELISA), as well as several novel immunochemical screening tests, have been developed. Most of these methods are very sensitive, specific, and simple to operate. Specific antibodies have also been used as immunohistochemical reagents and to arm affinity columns that are used as a cleanup tool for analysis of mycotoxins by other methods. Before selecting appropriate immunoassay for mycotoxin analysis, the following criteria should be considered: A.

Availability of Antibody and Markers

Both monoclonal and polyclonal antibodies against mycotoxins have been generated. Whereas most polyclonal antibodies were generated in rabbits, useful antibodies have also been obtained from eggs of hen, goat, and pigs as well as mice (28). Antibodies against different mycotoxins are summarized in Table 1 (38-90). Because most immunoassays for mycotoxins are based on the competition of binding between unlabeled toxin in the sample and labeled toxin in the assay system for the specific binding sites of antibody molecules, a well-labeled mycotoxin (as a marker) is needed in the assay system, in addition to a specific cmtibody. Approaches for the preparation of conjugates for antibody production and for the preparation of markers have been reviewed by the author (24, 26, 28).

B. Adequate Method for the Separation of Free and Bound Toxin For accurate quantification of the interaction between antigen and antibody, a good method for the sepnrntion of free and bound forms of toxin is important (28, 36).

C. Understanding the Specificity of the Antibodies Used in the Assay Depending on the approaches that have been used for raising antibodies, the degree of cross-reactivity (speciJiciv)of these antibodies with their respective structural analogs varies considerably; thus, one must be familiar with the specificity of the antibody to be used in the assay system (24, 26, 27). The cross-reactivity of antibody is determined by an immunoassay where various structurally related analogs of mycotoxin at a wide range of concentrations are used to compete with the binding of the marker ligand with the antibody in the assay. The concentration at 50% inhibition (IC5")of the binding is generally used as the basis to calculate the relative cross-reactivity for each analog. A typical example of such a competitive RIA and enzyme-linked immunoassay for aflatoxins is shown in Fig. 1. The cross-reactivity of various antibodies against mycotoxins is generally described alone in the publications for the production of specific antibodies against mycotox-

Chu

686

Table 1 AntibodiesAgainst

Mycotoxins3

Mycotoxins AAL toxin Aflatoxins: B1, B2, G1, G2 Aflatoxin metabolites: B2a. Q1, M1 aflatoxicol, DNA adducts Citrinin Cyclopiazonic acid Ergot alkaloids Fusarochromanone Fumonisin Kojic acid Ochratoxin A Patulin Paxilline related PR-toxin Rubratoxin B Secalonic acid Sporidesmin Sterigrnatocystin Trichothecenes: DAS, DON, FX. DOVE, AcDON, NIV, Roridin A, T-2 toxin, and T-2 toxin metabolites (HT-2, T-2-tetraacetate 3’-OH-T-2, dep-T-2) Versicolorin A Zearalenone a

Type PAb mAb, pAb mAb, pAb PAb mAb, pAb mAb, pAb PAb mAb, pAb PAb mAb, pAb PAb mAb, pAb PAb PAb PAb mAb, pAb PAb mAb, pAb

PAb mAb, pAb

References for most mycotoxins: see Chu(14,34,26,27,28). Addltlonal selected references: AAL toxin (38-40). AFB (41, 42). citrinin (43, 34).cyclopiazonic acid (CPA, 45-47), DON and acetyl-DON (48-54), ergot alkaloids (55-59), fumonisin (Fm; 37.60-70), fusarochromanone (71 ), OA (72-741, patulin (75). paxilline related (76-78). sporidesmin (77-80). sterigmatocystin (ST: Sl-83),nivalenol (84), T-3 toxin (85, 86). versicolorin A (S7). zearalenone (ZE: 85, 88-90). AF,aflatoxin: DAS. diacetoxyscirpenol:DON.deoxynivalenol;AcDON,Acetyl-DON:DOVE,deoxyverrucarol: FX, fusarenon-X: OA, ochratoxin A: NIV, nivalenol; dep-T-2, deepoxide T-3 toxin;mAb,monoclonalantlbody:pAb,polyclonal antibodies.

ins; several reviews have been appeared (24,26,36). Whereas antibodies against mycotoxins are very specific for each specific mycotoxin used in the immunizations, antibodies reactive with a group of mycotoxins, including aflatoxins and trichothecenes, have been also been made (91-96). D. Understanding the Effect of Matrix and Solvent System on the Assay Because there is always a possibility of the presence of some structurally related compounds in the sample that may react with the antibody, the sample Inatrix should be tested

Mycotoxin Analysis

687

LOG TOXIN CONCENTRATION (nglmL)

Figure 1 Examples of radioimmunoassay (RIA) and enzyme-linked immunosorbent (ELISA) of selected mycotoxins. (Top) The cross-reactivity of pAb with different aflatoxins using tritiated aflatoxin B1 as the marker. (Bottom) Typical standard curveof competitive-direct ELISA for selected mycotoxins and the respective mycotoxin-HRP conjugate was used as the marker in each case.

before the assay. In most of the immunoassays described below, sample cleanup is not necessary. Sample after extraction from the solid matirx could be directly used in the assay after appropriate dilution in the assay buffer. Nevertheless, the sensitivity increased after appropriate cleanup treatment.

111.

RADIOIMMUNOASSAY

The RIA procedure involves incubation of specific antibody simultaneously with a solution of unknown sample or known standard, and a constant amount of labeled toxin. After separation of the free and bound toxin, the radioactivity in those fractions is then determined. The toxin concentration of the unknown sample is determined by comparing the results to a standard curve that is established by plotting the ratio of radioactivities in the bound fraction and free fraction versus log concentration of unlabeled standard toxin.

Chu

688

A.

Principles

Radioimmunoassay involves the use of a radioactive marker, which competes with analyte in the sample for binding to an Ab. For RIA of large-molecular-weight antigen, either the antigen or antibody molecules can be labeled. It is also comtnon to use a radiolabeled second antibody, i.e., antibody against the primary antibody. In contrast, labeled mycotoxin is typically used in RIA of mycotoxins. Although RIA is simple and sensitive, it is limited by the need of a marker with high specific radioactivity, instruments for measuring radioisotopes, licenses for using radioactive materials, and disposal of radioactive materials. Because the radioactive marker has the same structural features as the compound to be analyzed, RIA provides good accuracy and is an effective method in the initial phase for screening of antibodies.

B. Preparation of Radioactive Ligands Used in the of Mycotoxins

RIA

In addition to the affinity constant of the Ab and Ag interaction, the specific activity of the radioactive ligand plays an essential role in the sensitivity of RIA. Although mycotoxins labeled with I4C, 'H, and '"1 have been used in RIA, the 'H-labeled toxins are most commonly used. Some high-specific-activity mycotoxins, including aflatoxin B 1(AFB 1) and ochratoxin A (OAj,are made commercially by a tritium exchange method (24). Other mycotoxins, including those in the trichothecene (TCTC) group, are made by reduction with high specific 'H-NaBH,. This was generally done by first oxidizing the secondary hydroxyl group and followed by reduction with'H-NaBH, (24j. Iodinated mycotoxin markers have also been made by first preparing a mycotoxin derivative containing a tyrosine, histamine, or tyramine. and then iodinating with "'1 using the standard methods such as Bolton-Hunter reagent, chloramine T, iodogen, lactoperoxidase, and iodo-beads, which are commercially available. For example, 3'-''sI-tyramine-AFB 1-0-carboxymethyl oxime (2300 Ci/nmol) was used in the RIA of both AFB 1 and AFM1 with high sensitivity (10 pg per assay) (31, 26, 32).

C. Separation of Ab-Mycotoxin Complex from Free Mycotoxins

Many methods have been used for the separation of the free and bound mycotoxins after incubation. In the earlier studies, methods such as equilibrium dialysis, amtnoniutn sulfate precipitation, precipitation with organic solvent, polyethylene glycol 6000, membrane filtration, dextran-coated charcoal, and albumin-coated charcoal have been used. As newer solid-phase matrix and more immunochemical reagents became available, antibodies could be coated noncovalently to a solid matrix such as polystyrene tubedbeads, microtiter plate, or modified nylon tubes or beads, or conjugated covalently to various matrices such as acetylbromo-cellulose, various types of Sepharose or agarose gels, controlled-size fineparticle magnetic gels, and others. Separation is achieved by filtration or centrifugation after the reaction. A second antibody has also been used to separate the free antigen and immunocomplex either by the formation of precipitate with the primary antibody or by coupling to a solid matrix (21, 26, 32).

Mycotoxin Analysis

689

D. Application of RIA for Mycotoxins Although RIA was developed in the early phase of immunochemical studies, this method is still used in some laboratories. The method is very simple. In general, the antibody either in the solid-phase or in solution is incubated together with the radioactive-labeled mycotoxin and the sample solution for an appropriate time and then one of the methods to separate the free and bound mycotoxin is used. The radioactivity in the solution, usually the free-form fraction, is then determined. RIA has been used for the analysis of AFB 1 in corn, wheat, peanuts, milk, serum, and eggs as well as for deoxynivalenol (DON) in corn and wheat, OA in serum and kidney, nivalenol in barley, PR toxin in cheese, and T-2 toxins in corn, wheat, serum, and urine (21, 23, 24, 28). Generally, RIA can detect 0.25-0.5 ng of purified mycotoxin in each analysis when tritiated mycotoxins are used as the markers. However, because of the sample matrix interference, the lower limit for mycotoxin detection in food or feed samples is about 2-5 ppb. Higher sensitivity, 0.0040.1 ng/assay, can be achieved by using iodinated-mycotoxin marker. The sensitivity of RIA can also be improved by a simple cleanup procedure after extraction and by using radioactive markers of high specific activity (24). As newer solid-phase matrices and more immunochemical reagents became available, more efficient methods for the separation of free and bound toxin were developed (24). Thus, separation can be achieved by a simple filtration or centrifugation step. A RIA-based immunoassay kit for mycotoxin analysis is commercially available (33, 96).

IV.

ENZYMEIMMUNOASSAY

A.

General Considerations and Assay Configurations

Enzyme immunoassay (EIA) is ageneral term for the immunoassays involving use of an enzyme as a marker for the detection of immunocomplex formation. Whereas the general principle of EIA is similar to that of RIA, there is an amplification system present in this assay, and thus, EIA is more sensitive. Since no radioactive substances are used, this assay avoids the problems encountered in handling radioactivity. Enzyme labeling can be done by conjugation of enzyme to Ag or Ab via the periodate oxidation and subsequent reductive alkylation method or by cross-linking using glutaraldehyde. Some of the methods used in conjugation of hapten to proteins can also be used. Although horseradish peroxidase (HRP) and alkaline phosphatase are the two enzymes most commonly used, others, such as glucose-6-phosphate dehydrogenase coupled with oxidoreductase and luciferase, glucose oxidase, beta-galactosidase, and urease, have also been used (24, 26, 28, 37). Depending on whether or not the immunocomplex is separated from the free Ag, EIA is further divided in two types. One type is a homogeneolrs system, which is based on modification of enzyme activity occurring when Ab binds with the enzyme-labeled Ag/hapten in solution. No separation is necessary in this assay. This system, which is also called ewyme multiplied im~nunonssny(EMIT), has been used for analysis of some antibiotics and hormones in theclinical diagnosis area. Because modification of enzymatic activity generally is not significant, this system is not very sensitive (pg/ml to mg/ml range) and has not been widely used in food analysis. The other is a hete~-ogeneoussystem involving separation of free and bound Ag-Ab. In this system, either Ag or Ab is bound to the solid matrix noncovalently or conjugated to it covalently. Unreacted Ab or Ag/hapten is easily removed by washing or centrifugation.

690

Chu

The term etz:yme-lird-ed i~nrr1ur~osol-bent assay (ELISA) is used for this type of assay and this system is widely used for analysis of a variety compounds, including mycotoxins. Solid phases such as tnicrotiter plates, cellulose, nylon beaddtubes, nitrocellulose membrane, polystyrene tubedballs, and modified magnetic beads have been used. In some cases, staphylococcal protein A or protein G is coated on the solid surface, entrapping the antibody for subsequent analysis. ELISA is further divided into two major types. One type is cotnpetitive ELISA (c-ELISA), which can be used for the analysis of both hapten and macromolecule; the other is noncompetitive sandwich ELISA, which is used only for divalent and multivalent Ag. c-ELISA is used most frequently for mycotoxin analysis; therefore, only this method will be discussed in detail. Depending on whether enzymelabeled Ag or Ab is used or whether Ab or Ag is coated to the solid phase, several types of competitive ELISA have been developed. Two major types, Le., direct competitive ELISA (dc-ELISA) and indirect conlyetitive ELISA (idc-ELISA), are used most commonly in mycotoxin analysis.

B. DirectCompetitive ELSA (dc-ELISA) In this assay, specific antibodies against mycotoxins are coated on the ELISA plate. The sample or mycotoxin standard solution is generally incubated simultaneously with enzyme-conjugate or incubated separately in two steps. The amount of enzyme bound to the plate is then determined by incubation with a chromogenic substrate solution. The resulting color/fluorescence, which is inversely proportional to the mycotoxin concentration present in the sample, is then measured instrumentally or by visual comparison with the standards. In this assay, the mycotoxin-enzyme conjugate (marker) and free mycotoxin compete for the same binding site on the solid-phase antibody. Although HRP is most commonly used as the enzyme for conjugation, other enzymes such as alkaline phosphatase and beta-galactosidase also have been used (14, 15, 21, 34, 26-28, 32-37). Excluding the time for sample preparation, dc-ELISA itself generally can be completed in 0.5-2 hr. In general, dc-ELISA is approximately 10-100 times more sensitive than RIA when purified standard is used and as little as 2.5 pgof pure mycotoxin can be measured. Since a cleanup step is usually not necessary, many samples can be analyzed within a relatively short period. dc-ELISA can detect 0.05-50 ppb of mycotoxins in foods and feeds (14, 15, 28). Like RIA, the sensitivity of ELISA is improved when a cleanup treatment is included in the assay protocol (24). Many examples could be cited. For example, Hongyo et al. (97) found a good correlation between the data obtained from a one-step ELISA of aflatoxin in corn with either HPLC or TLC, but the correlation between the ELISA data for AF in the mixed feed with HPLC and TLC was poor. In contrast, a good correlation was obtained when the mixed feed was subjected to column chromatography before ELISA. The efficacy of ELISA of fumonisin was also improved after a cleanup treatment of the samples (37, 63, 65, 98).We also found that the sensitivity of an ELISA of cyclopiazonic improved considerably when the samples were subjected to an immunoaffinity column (99). Owing to the use of better antibody and toxin-enzyme conjugates, the time required to run the ELISA has improved considerably. Thus, the entire ELISA procedure can be completed within 1 hr (24, 28, 33, 36, 37, 100). Better sensitivity can also be achieved by variation of substrate as well as by using amplification systems such as the biotinavidin interactions. For example, a more sensitive substrate, such as tetramethylbenzidine,

Mycotoxin Analysis

691

has been used for immunoassays using HRP as the marker enzyme (63) and fluorescent substrates have also been used to improve sensitivity. Thus, it is not surprising that some systems can detect as low as 0.05 pg in each assay. To save antibodies, Pesavento and Carter (101) have covalently conjugated antibodies against aflatoxin to the chemically activated hydrophilic membrane in an ELISA plate, which can be regenerated a number of times for repeated aflatoxin analysis. dc-ELISA is one of the most common protocols currently being used for immunoassay of mycotoxins. The sensitivity of dc-ELISA for mycotoxins in different commodities is summarized in Table 2 (102- 118). C.

Indirect Competitive ELSA (idc-ELISA or DoubleAntibody ELISA)

In the indirect competitive (idc-ELISA), a mycotoxin-protein (or polypeptide) conjugate is first prepared and then coated to the microtiter plate before assay. The plate is then incubated with specific rabbit (or other type) antibody in the presence or absence of the homologous mycotoxin. The amount of antibody bound to the plate coated with mycotoxin-protein conjugate is then determined by reaction with goat anti-rabbit (or anti-other type) IgG-enzyme cotnplex (which is commercially available) and by subsequent reaction with the substrate. Thus, toxin in the samples and toxin in the solid phase compete for the same binding site with the specific antibody in the solution. The idc-ELISA has also

Table 2 Sensitivity of Direct, Competitive ELISA for Selected Mycotoxinsa Standard Detection range limits Mycotoxins AAL AFB/AFs AFM CPA DON 3-AcDON 15-Ac-DON Fm H-Fm OA T-2 Type A TCTC ZE

Fooddfeeds (nglassay) C C, Wh, P, Pb M, Ch C, P, MF C, Wh B Wh C, MF, M, BR C Wh, B, K C, Wh C C

0.0025- 1 0.0012-1 0.1-0.6 0.012-125 0.0002-0.4 0.002-0.025 0.0005-50 0.1-15 0.025-0.5 0.0025-0.2 0.0025-0.2 0.02-2.5

(pg/kg) or (pg/l) 1-10 (1)b 0.10 (0.01) (0.05-0.1) 1000 (10) 16 50-100 10-500 (5-10) 30 (1-2) 2.5-50 (1) 50- 100 50 (10)

Data and references: see Chu (14, 27, 28). Additional selected references: AAL (40); AFs (102); AFM (103); CPA (104); DON (105-107); 3-Ac-DON (54); 15-Ac-DON (53); Fm (66, 67.70, 108-1 14); OA (1 15-1 17); ZE (1 18). Values in parentheses are for samples that had been subjected to a cleanup treatment before immunoassay. B, barley; BR, beer; C, corn; Ch, cheese; Fm, fumonisin; H-Fm, hydrolyzed Fm; K. kidney; M, milk; MF. mixed feed;P. peanuts; Pb, peanut butter; Wh, wheat;OA,ochratoxin A; Tctc.trichothecenes;otherabbreviations,see Table 1.

Chu

692

been widely used for analysis of a number of mycotoxins (14, 15, 21, 24, 27,28,36) with a sensitivity that is comparable to or slightly better than that of the direct ELISA in some cases. This type of ELISA requires less antibody (100 times less) and does not require preparation of a toxin-enzyme conjugate. However, it takes more analytical time (2 hr). To optimize the assay, selection of secondary antibody-enzyme conjugate in the idc-ELISA is important. In a recent study. for example, Okumura et al. (119, 120) found that their mAb-based ELISA for AFM1 was 50 times more sensitive in the HRP-labeled anti-mouse antibody than in the alkaline phosphate-labeled system. The sensitivity of idc-ELISA of ZE improved considerably when affinity-purified mycotoxin-conjugate was used as the coating antigen together with a flourescent substrate in the assay (118). To shorten the assay time for the idc-ELISA, two modifications were made by several investigators. One involved the conjugation of antibody to an enzyme, which is then used in the ELISA instead of a second antibody-enzyme conjugate, and the other involved premixing the antibody with the second antibody-enzyme conjugate before the assay (24, 36, 121, 122). The application and sensitivity of idc-ELISA for mycotoxins in different commodities are shown in Table 3 (123-128). D. Considerations of Sample Treatment in the ELISA In addition to the antibody affinity, efficacy of marker enzymes, enzyme substrate, and sample matrices, the presence of an extraction solvent system also greatly affects ELISA performance. Early studies showed that ELISAs could run in a system containing as much Table 3 Sensitivity of Indirect Competitive ELISA for Selected Mycotoxinsa ~~

Mycotoxins idc-ELISA AFs AFM DAS DON 3-Ac-DON Fm NIV OA ST T-2 HT-2 ZE Md-idc-ELISA AFB T-2

Foods/Feeds

C, P, Pb, F. Rs. S M c, w h c, Wh R C B B. K. MF, Wh

~

Standard limits Detectionrange (&assay)

M, S, U U C, W, MF

0.0002-1 0.0001-0.005 0.005-5 0.010-100 0.005-1 1-100 0.05-5 0.005-10 0.000 1-5 0.002-0.2 0.005-0.5 0.05-2.5

B, P. Pb, C B

0.01-1.5 0.05-5

s, u, w

~~~~

~

~~

( u g k g ) or ( u g L )

0.25-5 0.005 300 1000 (10) 1 200 (50) (30) 0.06-50 (1) 0.01-5 (0.05) 5 (0.2-1 ) (0.5) 1-60

Data and references: see Chu (14. 37, 28). Additional selected references: AF( 123-125); Ac-DON (54):FmB (68,136): ST (83): T-3 (127.128). Values i n parentheses are for samples that had been subjected to a cleanup treatment before immunoassay. B. barley; C, corn; Ch, cheese: F, figs; K. kidney; M, milk; MF. mixed feed; P, peanuts; Pb, peanut butter; R. rye; Rs, raisins; S, serum; Wh, wheat; U, urine; Md, modified; other abbreviations. see Tables 1 and 2.

Mycotoxin Analysis

693

as 20-30% of methanol (21, 24). Generally, samples contain 7-15% methanol in the phosphate buffer. In the ELISA of hydrolyzed fumonisin BI, Maragos and Miklasz (65) found that more dilution was necessary for corn samples extracted with acetonitrile than those extracted with methanol. This effect could be due to the solvent itself and also because more interfering materials were extracted by the acetonitrile. In a mAb-based ELISA for FmB1, we found that the presence of either 10% methanol or acetonitrile did affect the assay significantly (27, 28, 70). A number of studies have been carried out in recent years to investigate the efficacy of both direct and indirect immunoassays by comparing them with HPLC and TLC. Whereas good correlation has been found in most immunoassays (14, 21, 24, 28, 36), problems do exist for some other assays. For example, data obtained from immunoassay of Fnls were always higher than those obtained from chemical analysis (27, 112). This problem was attributed to the cross-reaction of the antibodies with some structurally related compounds. Once high-affinity antibodies became available, the nonspecific interaction was minimized (66-70). Collaborative studies for some ELISA protocols have been conducted. Several quantitative ELISA methods for the analysis or screening of mycotoxins have been adopted as first action by the AOAC (15-20).

V.

IMMUNOSCREENINGMETHODS

By shortening the incubation time and adjusting the antibody and enzyme concentrations in the dc- or modified idc-ELISA assay system, it is possible to do a quick screening test at certain toxin levels in less than 30 min (e.g., 20 ppb) (2, 14-16, 22, 24, 26, 37, 100, 130-134). Based on the same principle as the dc-ELISA, several other types of immunoscreening tests with sensitivity similar to ELISA were also developed. Rather than coating the antibody onto the tnicrotiter plate, the antibody is immobilized on a paper disk or other membrane (134-140), which is used directly as a strip (136) or mounted either on a plastic card (card screen test), a plastic strip (as dipstick; 141, 142), in a plastic cup (Cup test and Cite), or in a syringe (Cite probe and Idexx probe, 135). Antibody has also been coated on polystyrene beads (142, 143). The reaction is carried out on the wetted membrane disk. Thus, after reaction, the absence of color (or decrease in color), generally blue, at the sample spot indicates the presence of toxin in the sample. The reaction is generally very rapid and takes less than 10-15 min to complete. Like the above formats, dipstick-type enzyme imtnunoassay has been developed for quick screening of Penicillizm islnndicunz in rice grain (144) and 3-acetyl-DON (140) and T-2 toxin (145) in wheat. Abouzied and Pestka ( 146) immobilized different mAbs against AFB FmB and ZE, as multiple lines on the nitrocellulose strip and respective mycotoxin-peroxidase conjugates were used as the testing markers. Thus, this system can screen all three mycotoxins simultaneously with detection limits of 0.5,500, and 3 ng/mL for AFBl, FmB,,and ZE, respectively. Another screening test is the immunoaffinity method, which was originally designed for mycotoxins such as AF, OA, and ZE that fluoresce (14, 15, 21, 26, 28, 139, 147152). In this assay, sample extracts diluted in phosphate buffer are applied to the affinity columns in which specific antibody was covalently bound to the solid matrix. After washing to remove the unbound materials, specific mycotoxin is then eluted from the column with the appropriate solvent system and then subjected to other chemical analyses. For mycotoxins with native fluorescence such as AF, OA, and ZE, the toxin level in the eluate

Chu

694

could be directly determined fluorometrically or be determined after derivatization to enhance the fluorescence (150-1 52). For fumonisin and DON screening, it is necessary to introduce a fluorophore to the materials eluted from the immunoaffinity column (IAC) (27, 36). The sensitivity to the IAC screening tests for AFB 1, FmB 1, OA, and ZE is 2 ppb, 1 ppm, 5 ppb, and 0.2 ppm, respectively. The application of various immunoscreening tests to mycotoxins has been summarized by Chu (14, 21, 24, 27, 28) and Pestka (36), and most of the screening tests are commercially available as kits (2, 15, 22, 36). All of the rapid-screening test kits pemlit monitoring of mycotoxins semiquantitatively and have been found to be effective in screening mycotoxins in the field by FSIGS (2, 36, 154). Other evaluations for the commercial kits also concluded that such kits could be used for screening tests (17-20, 28, 90, 155-158). Collaborative studies for some of these immunoscreening tests have been done, and sotne of them have been adopted by the AOAC as first action for screening for AFs in different commodities (1 6-20, 28, 36, 138, 152, 157). With the increasing availability of commercial immunoassay kits, the AOAC International has established a research institute to evaluate the performance of different kits (16-20).

VI.

COMPLEMENTATION OF IMMUNOASSAYSWITH CHEMICAL METHODS

A.

lmmunoaffinityChromatography

With the availability of antibodies against various mycotoxins, imtnunoaffinity columns (IACs) were made by conjugating antibodies to a solid-phase matrix. These columns are then used either in a screening test as discussed above or as a cleanup column for subsequent chemical analysis (14, 21, 26-28). IAC was first used in the RIA (159) and later for recovery of AFM from urine and milk samples (160) for subsequent analyses. Although earlier applications of this technique were primarily aimed at biological fluids (149- 151, 161), the IAC has gained wide application as a cleanup tool for a number of mycotoxins and is not limited to fluid samples (14,21,26-28, 162). IACs for a number of mycotoxins are also commercially available. Table 4 summarizes the most recent applications of IAC technology to mycotoxin analysis. It is apparent that once the contaminants are removed by specific IACs, the solution can be directly subjected to liquid chromatographic (LC) quantitation, either off-line or on-line in an automated system, or by fluorometry. IAC not only serves as a cleanup tool but also concentrates the mycotoxin from a large amount of sample. Thus, it lowers the detection limits. Sometime as low as parts-per-trillion of mycotoxins can be measured. A number of collaborative studies indicate that IAC is an efficient method for cleanup of mycotoxins (14, 153, 162, 169, 170). A combination of an IAC column and PsCD is considered the official method for screening and clean-up of AF in several commodities (152, 162). Automation involving the use of an affinity column and HPLC was developed for routine analysis of AFM in milk (163-165, 204), AFB in peanut butter (171), in peanuts and corn (206), and in other nuts (207) and OA in cereal and animal products (192) and fumonisin (158, 187, 208). With the increased use of IACs as a tool for cleanup for mycotoxin analysis, one issue is the cost of the column. Research on regenerating columns for repeated use has also been conducted over the years; some of the approaches have been reviewed by Scott and Trucksess (162). Using IAC for cyclopiazonic acid as the tnodel, we found that the key to the successful regeneration of IACs

ed

Mycotoxin Analysis

695

Table 4 ImmunoaffinityChromatography of Mycotoxins AnalysisCommodities Mycotoxins AFM AFB AFB AFs AFL AFQ 1 AF-adducts AF-albumin CPA DON F I B . HFms OA OA ZE

ref. Md F. P Cff, P. nuts, figs, etc C.P,Ct,F,Pb,BR

Flh TLC HPLC/PsCD Fl/Br: HPLC/PCD

s, u

HPLC HPLC HPLC, ELISA HPLC, ELISA ELISA HPLC HPLC: LC/MS HPLC CE ELISA, HPLC, MS

U TAU S C, P, feed

c. w C, starch Cff, T. W. C, So Cff, c , so M, u, c

149, 163, 164. 204 166, 167 168-174 150,152,154, 174. 175 176 177 161, 178-182 182- 186 99 256 158,187-190 173, 191-196 197 198-203

Commodities tested: BR, beer. Cff, coffee; T, animal tissues: So, sorghum; other abbreviations, see Tables 1 and 3. Methods for final analysis: FI/Br and F1 represent fluorometric analysis of the solution eluted from the column with and without treatment with bromine solution, respectively.

rests on the equilibration time. One must equilibrate the IAC in the loading buffer for sufficient time (>4 hr) before reuse (99).

B. Combination of Immunoassay with Other Chemical Methods 1. Combination with HPLC and TLC With the availability of sensitive ELISA methods, this technique has proved effective as a postcolumn monitoring system for HPLC (209). This is especially useful for the analysis of compounds with no specific absorption, such as TCTCs and Fms. For example, in the analysis of various type A TCTC mycotoxins, the sample extract with no cleanup treatment was first subjected to HPLC with a C-18 reverse-phase column. Individual fractions eluted from the column were analyzed by ELISA using “generic” antibodies against group A TCTCs. This approach can not only identify each individual group A TCTC, but can also determine its concentration quantitatively. As little as 2 ng of T-2 toxin and related TCTCs as well as their metabolites can be monitored by this method. A combination of HPLC and ELISA technology proved to be an efficient, sensitive, and specific method for the analysis of TCTC (2 10-212) and other mycotoxins (8 1, 82, 2 13). In a recent study, we were able to determine FrnBl, FmB2, Fr-3, and AAL toxin TA simultaneously when two types of pAb, one against Fms and one against AAL toxins, were used in the postcolumn ELISA (40). The detection limit was 0.1 ng FmB 1 per tube (0.5 ml). Recovery of FmB1 added to ground corn in the 100-1000-ng/g range in this system was 78.8%. Analysis of extracts from cultures of three Alternaria nlternatu strains revealed that both FmB 1 and the AAL toxin TA were present, but their amounts varied considerably with the cultures tested.

Chu

696

Likewise, immunoassay has been used in combination with TLC (213) in which the crude sample extract was applied to the TLC plate. After separation, each fraction in TLC was analyzed immunochemically. It is interesting that while this approach quantifies the known toxins, it is also capable of uncovering new mycotoxins with structures similar to those of the known mycotoxin. Miles et al. (214) were able to isolate paxinorol, a new paxilline derivative, when they monitored the TL chromatogram with ELISA using antibody specific to paxilline. 2. Combination of High-Performance TLC (HPTLC) with lmmunoblotting An approach called HPTLC-ELISAgram was introduced by Pestka (215). This method involves separation of mycotoxins using HPTLC, followed by blotting the chromatogram onto a nitrocellulose membrane coated with antibody, incubation with mycotoxin-enzyme conjugate, and a final incubation with substrate to develop the color. Although this method has good sensitivity, the need for a large amount of antibody limits its wide application. 3. Combination of Immunofluorescence and Capillary Electrophoresis (CE) Based on the competition between unlabeled FmB 1 (i.e., from a sample) and a fluoresceinlabeled FmB 1 reagent (FmB 1-FL) with the mAb, Maragos (2 16,217) used CE to separate the bound and free FmB 1-FL. In the assay, purified FmB 1-FL was subjected to CE. Addition of purified mAb to FmB 1-FL before separation resulted in the formation of mAbFmB 1-FL complex with resulting quenching of fluorescence and decrease in the intensity of the FmB1-FL peak. When a sample containing FmB 1 is present, it competes with FmB 1-FL for binding of the limited amount of mAb causing an increase in FmB 1-FL peak. The intensity of such increase is directly proportional to the amount of unlabeled FmBl present. The IC5" of unlabeled FmBl was highly dependent upon the antibody concentration and ranged from 58 to 4170 ng/ml (at 15-75 pg/ml of antibody). The method is rapid and requires only 6 min for cotnplete analysis of FmBl standard.

VII. A.

OTHERNEWLYDEVELOPEDIMMUNOCHEMICAL METHODS Anti-idiotypeAntibody-BasedImmunoassays

The development of immunochemical methods for tnycotoxin detection has led to a great demand for specific antibodies and related immunochemical reagents for the assay. An alternate approach to preparing imtnunochemical reagents is through generating anti-idiotype (anti-ID) antibodies (218). Anti-ID (Ab2j for large molecules have been well developed and have been applied to clinical diagnosis and ilnnlunotherapy (218). Recent studies have succeeded in generating Ab2 against a number of small-molecular-weight haptens, including mycotoxins AFs (219-221), FmB 1 (98), and T-2 toxin (222,223).Anti-idiotype antibodies for mycotoxins were demonstrated in different animal species after immunization with the affinity-purified original idiotype antibodies (Abl) (98, 219j. Ab2 were not only bound specifically to the original Ab1 but also were capable of being used as an immunogen in generating anti-anti-ID antibodies (Ab3). Whereas Ab2 could not be used as a mycotoxin-protein conjugate in the indirect ELISA for the determination of aflatoxin and T-2 toxin, an Ab2-based indirect ELISA has been established for Fm analysis (98).

Mycotoxin

Analysis697

Thus, these anti-idiotype antibodies are indeed the surrogates of mycotoxins. Most recently, a hybridoma cell line that generates monoclonal Ab3 was obtained in our laboratory (257). From the ID5"values, it is apparent that the Ab3 have similar characteristics to the original Ab 1. The availability of Ab2 and Ab3 for mycotoxins has provided a new generation of immunochemical reagents, which could be used for both therapeutic and analytical purposes. Ab2, a surrogate of the toxin, could be used as the immunogen in generating antibodies for the toxin (thus. a vaccine) and could also be used in the immunoassay. In an in vitro study of the effect of antibodies on the binding of aflatoxin to DNA, Hsu (224) found that Ab2 were capable of inhibiting the binding of aflatoxin B, to DNA, but the inhibitory effect was not as high as that of the Abl. 6. Immunoassay for Mycotoxin-ProducingFungi Other than mycotoxins, antibodies against specific fungi (144, 225-228) as well as several enzymes involved in the biosynthesis of aflatoxin(229-234) and trichothecenes (235,236) have been generated also. Five mAbs against Aspergillus~nvuswere recently produced by Candlish et al. (237) and those mAbs were used in the immunoassays for identification of A. flavus. Whereas we used the partially purified proteins for generating of antibodies against the enzymes in the aflatoxin biosynthetic pathways (229-234), Shapira et al. (238) used both the culture filtrate and two chimeric proteins, which were expressed in Escllerichin coli from genes ver- 1 and npn-2, as the antigens for generating the antibodies against toxic Aspergillus pnrnsiticus and A. .fkrvus. The pAb generated from the chimeric proteins were also specific for these fungi. These antibodies produced in different laboratories have been used for identifying specific fungi in foods, for studying the kinetics of enzymes involved in the biosynthesis of mycotoxins, and for cloning the genes that encode the enzymes for mycotoxin synthesis.

C. Development of Biosensors Although development of An/Ab-based biosensors for mycotoxin detection was initiated in the late 1980s, application of this technology only emerged in recent years. Nevertheless, some of the principles and methodology are useful for designing the biosensors. In the so-called "hit-and-run" assay (239,240) for T-2 toxin, a T-2 toxin column was equilibrated with a fluorescein isothiocyanate (F1TC)-labeled Fab fragment of IgG (anti-T-2 toxin). Samples containing T-2 toxin were injected into the column. The FITC-Fab that eluted together with the samples containing T-2 toxin was then determined in a standard flowthrough fluorometer. A similar approach in which ribonuclease-labeled Fab was used as the indicator was also reported (240). Another approach that may lead to the development of a biosensor is a homogeneous immunoassay for T-2 toxin, which involves the use of liposomes and complement (241). Whereas these methods were not as sensitive as the regular ELISAs, several biosensors with some modifications can be used for rapid screening of mycotoxins. 1. Time-Resolved Fluoroimmunoassay (FI) This method involves the use of Europium ion (Eu)-labeled antibodies and has a sensitivity similar to that of most ELISA methods with an ICsovalue of 0.2 ng AFB l/ml (123, 242). The same approach can be used for other mycotoxins.

698

Chu

2. Fiberoptic lmmunosensor In this assay, mAb are covalently bound to an optical fiber and an evanescent wave effect was utilized to excite the flourescent-tagged toxin near the surface of the mAb-fiber as the tagged toxin bound to the fiber. In the presence of unlabeled toxin, it competes with the labeled toxin for binding with mAb and results in a decrease in signal. Thus, it is also a competitive assay and has been tested with T-2 toxin (243) and fumonisin (244). Using mAb against FmB 1 and FnlB 1-FITC, Thompson and Maragos (244) tested the feasibility of this system for analysis of FmB 1. The assay involves: (1) saturation of mAb binding sites by FmB 1-FITC, (2) competition of FmB 1 and FmB 1-FITC with displacement of the labeled toxin, and (3) resaturation of binding sites with FmB 1-FITC. The signal generated in the assay was inversely proportional to the concentration of FmB 1. This sensor has a working range of 10- 1000 ng of FmB l/ml, an IC5,)of 70 ng/ml, and a limit of detection of 10 ng/ml. These values compared favorably with those for currently available ELISA techniques. The methanol/water-extracted corn sample did not affect the sensor performance. 3. Automated Particle-Based lmmunosensor (API) This sensor is based on the kinetic exclusion assay. The system consists of a simple fluorimeter with a 1.5-mm-diameter glass capillary to serve as the flow cell within the final lens. Application of this system for the analysis of AFBl was studied by Strachan et al. (245). In the assay, the appropriate amount (ca. 100 beads) of polymethylmethacrylate beads (98 pm) coated with AFB1-BSA is pumped into the capillary and trapped on a filter. The sample or calibrated standard solutions that had been incubated with antibodies were then allowed to pass through for a period of 120 sec, followed immediately with FITC-labeled goat-anti-rabbit antibody (130 sec), and then finally washed with buffer to remove excess label. The fluorescence during each step of the reaction was recorded. The amount of antibody to the sample can be calculated by measuring the difference in voltage from the sensor during each step and at the end of the assay. This automated sensor can easily detect AFBl down to a level of 4 ng/g (4 ppb) in reference food materials in 8 min excluding the sample extraction step (