Table Of ContentSYMPOSIUM ON MYCOTOXINS
IN HUMAN HEALTH
SYMPOSIUM ON
MYCOTOXINS
IN HUMAN HEALTH
The Proceedings of a Symposium held in Pretoria
from 2nd to 4th September 1970 under the auspices
of the South African Medical Research Council
with the collaboration of the South African
Council for Scientific and Industrial Research.
Edited by
I. F. H. PURCHASE
Palgrave Macmillan
ISBN 978-1-349-01320-3 ISBN 978-1-349-01318-0 (eBook)
DOI 10.1007/978-1-349-01318-0
©South African Medical Research Council1971
Softcover reprint of the hardcover 1st edition 1971 978-0-333-13146-6
All rights reserved. No part of this publication
may be reproduced or transmitted, in any form
or by any means, without permission
FIRST EDITION 1971
Published by
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SBN 333 13146 0
CONTENTS
page
Introduction vii
Introductions to Papers xii
BIOCHEMISTRY SESSION
Effects of Aflatoxins on in vivo Nucleic Acid Metabolism in Rats 1
The Metabolism and some Metabolic Effects of Sterigmatocystin 11
The Effect of Aflatoxins on Pancreatic Deoxyribonuclease 19
The Effect of Aflatoxin Bh Aflatoxin Bz and Sterigmatocystin on
Nuclear Deoxyribonucleases from Rat and Mouse Livers 31
Aflatoxin Metabolism 47
Biochemical Studies on Ochratoxin A 53
Porphyrin Metabolism in Primary Hepatoma 65
Surveys for Alpha-feto-protein among Bantu Goldminers 71
MYCOLOGY SESSION
Mycologic and Mycotoxic Examination of Cycads 75
Field Survey of Mycotoxin-producing Fungi Contaminating
Human Foodstuffs in Japan, with Epidemiological Background 101
(I) Mycological and Chemical Aspects of the Detection of
Mycotoxin Producers 101
Isolation of Aspergillus ochraceus producing Ochratoxins from
Japanese Rice 107
Production of Citreoviridin, a Neurotoxic Mycotoxin of
Penicillium citreo-viride Biourge 115
Storage Surveys and how they may be used both to Detect and
Estimate Fungal Contamination in the Diet 133
TOXICOLOGY SESSION
The Toxicology of Aflatoxin 141
Hepatic and Renal Pathology Induced in Mice by Feeding
Fungal Cultures 153
Toxicological and Biological Properties of Fusarenon-X, a
Cytotoxic Mycotoxin of Fusarium nivale Fn 2B 163
v
Field Survey of Mycotoxin-producing Fungi Contaminating
Human Foodstuffs in Japan, with Epidemiological Background 179
(II) Biological Effects of the Mycotoxins Produced by the Fungi
Isolated from Foodstuffs 179
Experimental Evidence that Lupinosis of Sheep is a Mycotoxicosis
Caused by the Fungus Phomopsis leptostromiformis (Kiihn)
Bubak 185
Aflatoxin Carcinogenesis in Rats: Dietary Effects 195
The Acute and Chronic Toxicity of Sterigmatocystin 209
The Effects of Aflatoxin B1 and Sterigmatocystin on Two Different
Types of Cell Cultures 215
Recently Discovered Metabolites with Unusual Toxic
Manifestations 223
EPIDEMIOLOGY SESSION
Epidemiological Interactions 231
Dietary Aflatoxin Loads and the Incidence of Human
Hepatocellular Carcinoma in Thailand 245
Preliminary Results from Food Analyses in the Inhambane Area 263
Aflatoxin Ingestion and Excretion by Humans 271
The Incidence of Fungi in Foodstuffs and their Significance, based
on a Survey in the Eastern Transvaal and Swaziland 281
Biological Screening as a Laboratory Aid in Determining Cancer
Aetiology 291
Author Index 299
Subject Index 301
vi
INTRODUCTION
It was towards the end of the last century that scientists found that certain
products of micro-organisms were responsible for various diseases in both
man and animals. Further research during this century has increased the
knowledge in the field of microbial toxins enormously and this is particu
larly so of bacterial toxins. We are in a position today of knowing not only
the cause of these diseases, but also how to treat and prevent such diseases
as botulism and salmonella, streptococcal and staphylococcal food
poisoning. The knowledge of mould-induced food-poisoning is much more
scanty and research on causation and prevention of mould intoxication
has far to go.
It is interesting to note that, although the general knowledge of mould
toxicoses is limited, one of the oldest food-borne diseases recognised was a
mould infection. Ergotism, known a thousand years ago as St Anthony's
fire, was a disease which killed many thousands of people in Europe. This
disease is caused by the fungus Claviceps purpurea which grows on rye.
The rye grain becomes completely replaced by selerotia of the fungus and
turns black. Ingestion of between 1 and 1·5 g of diseased rye grains daily
can result in symptoms. Two types of the disease occurred, one in which the
main syndrome was gangrene of the extremities caused by peripheral
thrombosis of the arteries, and the other in which the syndrome was due
to damage to the nervous system resulting in numbness, twitching, con
vulsions, blindness, deafness and paralysis. It was not for hundreds of years
that the discoloured grain was recognised, as being the cause of the disease,
by Kaspar Schwenckfeld in 1600. The disease continued to occur right into
the 19th century in Europe and America and was last recorded on a large
scale in Russia in the 1920s and 1930s.
More recently a disease known as alimentary toxic aleucia (A.L.A.) was
recorded in the 1940s in Russia. In 1944 this was particularly severe in the
Orenburg district where 10% of the population suffered from the disease.
As the name suggests, the patients suffer from haemorrhagic diathesis,
leucopenia, agranulocytosis and also necrotic skin lesions. There was
widespread mortality at the time in all age groups but more particularly in
malnourished people. The investigation of the disease showed that it was
caused by mouldy grain. At first millet was thought to be the only source
of infected material but later wheat and barley were shown to cause the
disease. The conditions under which the disease occurs illustrate vividly
that fungi can grow under the most unusual conditions. Three fungi were
vii
viii INTRODUCTION
shown to be most important in causing the disease (Fusarium pose,
Fusarium sporotrichoides and Cladosporium epiphyleum) and extensive
laboratory investigation showed that these fungi produced their toxins
only when the temperature dropped below freezing point. Under these
conditions, although mycelial growth was limited, sporulation was profuse
and the steroid-like toxin was produced. These facts fitted in well with the
factors known to lead up to outbreaks of the disease. During the war years
manpower was not always available to reap the harvest and as a result it
was left standing in the fields during the winter. It was during this over
wintering that the fungus grew, became toxic and subsequently caused the
epidemic in Russia.
Of course, animals are, in general, more likely to suffer from myco
toxicoses because of the way in which they are fed. This is reflected in the
large number of diseases which are recognised in animals. Fungi are known
to produce 'haemorrhagic syndrome' in poultry, nephrosis in pigs,
stachybotrio-toxicoses in horses and many other diseases. Recently South
African research workers were able to identify the fungus responsible for
Lupinosis in sheep, a disease which has for many years been suspected of
being a mycotoxicosis. All attempts to identify the fungus have been
unsuccessful until recently and I am very pleased to see that a report
on this most interesting syndrome is to be delivered at this symposium.
In spite of the fact that the major epidemics caused by mycotoxins
occurred in Europe, it is no mistake that this symposium on mycotoxins in
human health is being held in South Africa. Research in the last decade
has been carried out by the CSIR and the newly formed MRC and I am
not being unduly biased in saying that this South African research effort
has contributed significantly to the basic knowledge which has been so
carefully gathered. We are, however, very lucky to have such a large and
distinguished overseas contingent at this symposium.
Japanese research workers have been involved in mycotoxin research for
many years and their work on 'yellowsis' rice in the 1940s can probably
be considered the forerunner of the work on carcinogenic mycotoxins.
Penicillium islandicum later isolated from mouldy rice in Japan yielded
toxins which have proved to be hepatocarcinogens in mice. This was truly
an astounding discovery and it has been acclaimed as a fine piece of research
work by all who know the field.
It is strange that the Japanese discovery had so little impact on research
in the Western world. In spite of the fact that this work was published in
the early '50s it was not until 1960 that work on other carcinogenic
mycotoxins was undertaken. Most of this work occurred as a result of the
so-called 'Turkey-X' disease which killed thousands of turkeys in Britain.
The cause of the mortality was traced to a batch of Brazilian groundnut
INTRODUCTION ix
meal which contained a blue-fluorescent substance. This substance was
later shown to be produced by the mould Aspergillus flavus and hence was
given the name aflatoxin. It was found that several domestic animals
including poultry, pigs and cattle, were susceptible to the toxin. Labora
tory studies were soon initiated on the toxic effects of aflatoxin and it was
found to be not only a hepatotoxin but also a potent hepatocarcinogen,
producing liver tumours in rats, trout, ducks and pigs. This discovery,
which resulted directly from the mortality in poultry, turned out to be the
forerunner of a vast amount of research. It proved to be an important tool
in studying the biochemical events leading up to cancer induction. The
concept that a fungus could produce a metabolite which was a carcinogen
stimulated numerous people for the first time to suggest that mycotoxins
could be involved in the aetiology ofliver cancer, in spite of the fact that a
similar concept had been formulated in Japan nearly 10 years earlier.
Impetus was given to this idea by subsequent findings which indicated that
aflatoxin was not only a carcinogen, but that it was the most potent
carcinogen known to man. A few hundred microgrammes are enough to
produce tumours in a high percentage of test animals.
The suggestion that mycotoxins could be responsible for human liver
cancer opened up a whole new field of investigation. The concept that
mould-produced toxins could cause chronic disease in man was the major
advance in thought, and it proved to be the trigger which has unleashed an
avalanche of publications in this field until there are now nearly 1000
publications just on this subject.
In the particular context of human disease in South Africa, the idea that
fungi might be responsible for chronic disease, and particularly liver cancer,
received enthusiastic support. Liver cancer, like so many other cancers,
has a variable distribution through the world and this variation suggested
that it must be caused by an environmental agent. Many suggestions had
been made which connected such diverse agents as virus infection,
bilharzia and malnutrition with the high incidence of the disease. In each
case, however, there was evidence which suggested that a direct causal
relationship between these agents and the disease in man was unlikely.
Mycotoxins appeared to be much more likely than any of the previously
suggested agents to cause the disease. It was known that liver cancer
occurred with high incidence in areas of high humidity and temperature
and in relatively primitive populations. Fungi require high temperatures
and humidity in which to grow and would grow in foods that were stored
under primitive conditions. It seemed, therefore, that the ingestion offood
contaminated with mycotoxins could provide a plausible explanation for
the high incidence of liver cancer in certain areas in Africa.
There are a number of factors which make further study of this hypo-
X INTRODUCTION
thesis uniquely difficult. In all the examples that I have mentioned of
mycotoxins being responsible for a particular human or animal disease,
the disease has been relatively acute with a short latent period between
ingestion of the toxin and the development of lesions. It is, therefore,
relatively easy to connect the development of the toxicity with the induction
of the disease. In the case of liver cancer it is much more difficult. The
latent period between ingestion of the toxin and development of the
disease may be relatively long. Other toxin-induced cancers, such as bladder
cancer resulting from industrial exposure to a carcinogen, have latent
periods of 10-40 years. There is no reason to believe that liver cancer
behaves differently, although in high-incidence areas children under the
age of 10 do contract the disease. We are thus faced with the problem of
identifying a toxin in the diet which is going to produce disease one or
more decades hence.
A further complication is the rather variable way in which fungi produce
their toxic metabolites. The acute diseases give us a good example of this
variability. Ergotism occurred in isolated outbreaks, for example, that
occurring in A.D. 943 in France which killed thousands of people. Similarly
A.L.A. had a variable incidence in Russia and only occurred after tem
peratures had reached freezing point. The animal diseases, such as facial
eczema in New Zealand, also had a very patchy and variable incidence.
Aflatoxin itself is another good example. Studies by agriculturalists in
groundnut-producing countries, including South Mrica, have shown that
the incidence of contaminated batches of groundnuts may vary from 0 to
over 40% from one year to the next. In general the higher incidence occurs
in drier years-a seeming paradox as the fungus requires high humidity in
which to grow. The explanation is that in drier years insects damage the
nuts with the result that a micro-climate is created in the damaged nuts
which is conducive to mould growth. From these examples it can be seen
that mycotoxins have a habit of behaving in a rather unexpected way which
tends to result in marked variations in the amount present in a given food.
These two factors, namely a long latent period and a variable level of
toxin in diets potentially infected with moulds, provide an extremely
complicated background to the mycotoxin hypothesis. What at first
appeared to be relatively simple now appears to be complicated and
unfortunately vague. The hypothesis will, in fact, be extremely difficult to
prove. One of the key questions to which there is as yet no answer is
whether man himself is susceptible to the chronic or even acute effects of
aflatoxin or other mycotoxins. In the absence of a direct answer to this
question, circumstantial evidence will have to be accumulated to give us an
idea of the susceptibility of man to these toxins. Careful studies are required
which will measure the incidence of liver cancer and the intake of myco-
