First Botany 135 Exam, October 11, 2005-Mycotoxins

[Guest guest]

This primarily deals with ingestion, but it contains alot of

interesting information concerning mycotoxins.

KC

First Botany 135 Exam, October 11, 2005

http://www.botany.hawaii.edu/faculty/wong/BOT135/lect11.htm

Mycotoxins

Introduction

Fungal diseases are common place in plants and animals. In such

diseases, the fungi are actively growing on and invading the body of

their hosts. There is another means by which fungi can cause harm

without invading our bodies. When fungi grow on a living organism or

on stored food material that we consume, they may produce harmful

metabolites that diffuses into their food. It is believed that fungi

evolved these metabolites as a means of protecting their food supply

by preventing other organisms from eating it. These metabolites are

referred to as mycotoxins, which literally means " fungus poisons " .

Fungi that produce mycotoxins do not have to be present to do harm.

If a fungus was growing in, say a grain storage silo, the

environment may have become unsuitable for the fungus and it dies.

Even though the fungus is no longer alive, while it was growing, if

it produced a mycotoxin, it will have poisoned the grains. So for

those of you who are always looking to save a little money by buying

cheese that has been contaminated with a fungus and cutting out the

part where the fungus is growing, perhaps this is not such a good

idea. It is possible that the fungus growing on your cheese has

produced a mycotoxin that has diffused throughout the cheese, even

though the fungus itself has not. The effects of poisoning by

mycotoxin is referred to as mycotoxicoses. The knowledge that

mycotoxicoses is the result of fungal actions was a relatively,

recent discovery. This is understandable since illnesses in this

case is due to consumption of mycotoxins that has been released by

the fungus and is not directly caused by the fungus. So

demonstrating this would not have been an easy task.

We now know that many species of fungi produce mycotoxins, but why?

It is thought fungi have evolved production of various mycotoxins in

order to prevent other fungi or animals from consuming " their " food.

By secreting their mycotoxin into their food the fungus will inhibit

growth of other fungi and discourage rotten or other small animals

from eating their food.

We will have several lectures on mycotoxins. Today, we will cover

some of the more common mycotoxins that are produced by by molds

growing in food and describe their symptoms. The following two

lectures will be concerned with a number of different toxins that

are all derived from Claviceps purpurea, the fungus responsible for

the disease on rye, commonly referred to as Ergot, and later in the

semester we will talk about mushroom toxins.

Early Attempts to Demonstrate the Existence of Mycotoxin

The existence of mycotoxins was not documented until 1960. However,

just as in the case of diseases, the concept that moldy food could

lead to illness in people or domestic animals was long suspected

before their existence was demonstrated by science. It is a greater

problem, presently, than it was in the distant past. Long ago,

before there was adequate means of long term storage for perishable

goods, food was normally consumed a short time after it was

acquired, but as the world has become more industrialized and

technological advanced, storage of food has become more of an issue.

Food is now commonly stored for long periods of time, giving fungi a

greater opportunity to contaminate our food.

Before 1900, in Italy, researchers there believed consumption of

moldy corn by children led to the development of illness

(Christensen, 1975). Some experiments, done at that time, included

the isolation, and growth of the suspected fungus in pure culture,

and isolation of toxic compounds from the fungus that the

researchers believed to be the cause of the illness. However, since

the compound was not identified and was not actually isolated from

the moldy corn, it could not be concluded that this compound was the

cause of the illness or that the compound in question was even

present on the moldy corn. Nevertheless, it appeared that there was

a correlation between the illness and consumption of moldy corn, but

this did not eliminate the possibility that it was the fungus,

itself, that caused the disease, which most people believed to be

the case. It was also possible that there were other reasons for the

illnesses that were observed.

Burnside, et al (1957) studied an extensive outbreak of moldy corn

disease in the southeastern United States in the early 1950's where

hundreds of wild pigs foraging in cultivated corn fields became ill,

and many died. Teams of veterinarians and mycologists collaborated

to determine the cause of the deaths of these pigs. They isolated a

number of different fungi from the moldy corn and inoculated each

fungus on moist corn that had been sterilized and then fed them to

pigs. The consumption of corn inoculated with Aspergillus flavus

caused outward signs and inward lesions found in other cases of the

so-called moldy corn disease. However, since there was no toxin(s)

isolated, there was little attention paid to the article since it

still seemed like old news, i.e. domestic animals poisoned by eating

moldy corn.

It would not be until 1960, when approximately 100,000 turkeys and a

lesser number of other domestic birds died in England, causing

losses of approximately several hundred thousand dollars, before the

first mycotoxin was isolated and identified. As you might guess,

this did not happen immediately. Initially, the disease was thought

to be caused by a virus and the syndrome was named " turkey-X

disease " . The " X " here indicated that the cause of the disease was

unknown. However, with a great deal of detective work, on the parts

of the researchers, soon the cause of the disease was traced to feed

that was produced by Oil Cake Mills, Ltd. (research always seems to

get done more quickly and receive more priority when loss of large

sums of money is involved). The oil cake feed was composed mostly of

peanuts. However, it seemed unlikely that the peanut meal itself was

toxic, since peanut meal had long been used as a feed ingredient and

was known to be an excellent source of protein. Thus, it was

reasoned that something must have been added to the peanut meal to

make it toxic, and one possibility that was investigated was that

peanuts had been made toxic by fungi growing in them. From their

isolations, the investigators identified Aspergillus flavus, the

same fungus that was isolated by Burnside and his research teams.

The isolated fungus was again inoculated into the feed and fed to

the turkeys. Shortly after feeding, the turkeys died with external

signs and internal lesions identical to those observed in the birds

that had previously died in the field.

Unlike Burnside, however, chemists were also employed in this

investigation, and they were able to isolate and identify the toxin

from the oil cake feed. The mycotoxin isolated was named aflatoxin,

the " a " from Aspergillus and " fla " from flavus. Feeding test of food

containing aflatoxin, with various laboratory animals, demonstrated

that to varying degrees, all animals tested were sensitive to

aflatoxin. Even consumption of extremely small amounts of aflatoxin

damaged various internal organs and could induce development of

cancer to the liver.

This was of great concern among the nutritionists and those

concerned with problems of pubic health, e.g. The Food and Drug

Administration. There was great concerned domestically since peanuts

and peanut products were/are of economic importance. It was also of

international significance, since peanuts at that time was being

lauded as an excellent source of protein, for developing countries,

by UNICEF (United Nations International Children's Emergency Fund)

and other such organizations. Deficiency in protein often results

in " kwashiorkor " .

Kwashiorkor (kwä´shê-ôr´kôr´), protein deficiency disorder of

children, prevalent in overpopulated parts of the world where the

diet consists mainly of starchy vegetables, particularly Africa,

Central and South America, and S Asia. Such a diet is deficient in

certain amino acids, which make up proteins vital for growth.

Depending on the extent, onset, and duration of the deficiency,

manifestations include skin changes, edema, severely bloated

abdomen, diarrhea, and generally retarded development. The Concise

Columbia Encyclopedia is licensed from Columbia University Press.

Copyright © 1995 by Columbia University Press. All rights reserved.

Other characteristics include anemia, depigmentation of the skin,

and loss of hair or change in hair color. Usually, occurring in

children, shortly after weaning. Peanut products were developed in

various forms, especially in tropical and subtropical countries, for

general distribution. Were these people now exchanging kwashiorkor

for potential risk of liver damage and cancer from consuming

aflatoxins? Thus, there was a great deal at stake, which provided an

impetus to act on this matter, immediately. In the United States, as

soon as awareness of aflatoxins surfaced in the 1960s, programs were

established by peanut growers, aided by concentrated research, to

reduce the possibility of aflatoxin occurring in edible peanuts and

peanut products. That they have been successful is indicated through

random sampling, by the FDA, of only an occasional batch of peanut

products that contains aflatoxin. What has been discovered have

almost never been found in sufficient amount to be of any real

concern. However, in other countries, in which there are no such

regulatory bodies, the people undoubtedly consume an appreciable

amount of aflatoxins.

The Fungus

Aspergillus flavus is actually not a single species, but a " species

complex " , made up of eleven species that are known to occur in many

kinds of plant materials, including stored grains. One of the

species in the complex, A. oryzae has long been used in the Orient

to prepare various kinds of food products, such as sake, tofu and

soy sauce, which in turn is used in the United States. Were

aflatoxins present in these products as well? This was a question

that needed to be answered. Research began to take place at a rapid

pace and continues to do so. The number of papers published that

have to do with aflatoxins number in the hundreds annually.

What was determined in early research of aflatoxins is that the

conditions which allows for growth of A. flavus and aflatoxins is

very narrow. Aspergillus flavus seldom invades stored grains alone,

i.e. as a pure culture. Various other species of fungi will normally

grow on a substrate prior to invasion by A. flavus, e.g. A. glaucus

and Candida pseudotropicalis. In a preinvaded substrate, regardless

of how dense the A. flavus invasion may be, aflatoxin will not form.

This was demonstrated at the University of Minnesota where A. flavus

as well as other fungi were grown on grain at moisture content and

temperature range that were optimal for aflatoxin production.

Although, there was a very dense mycelial growth of A. flavus, the

grain that was fed to the various kinds of experimental animals,

ducklings, white rats and baby chicks, for as long as eight months,

there was not a single case of death from consumption of the feed.

In fact, weight gain was the same as that of those animals not fed

the with grain containing the fungal growth. Thus, the amount of

mycelial growth that occurs in animal feed, with several species of

fungi involved, even if one is known to be an aflatoxin producer,

was apparently safe to consume. Thus, in order for aflatoxin

formation to occur in say a storage bin full of peanuts, A. flavus

must be growing alone and the peanuts cannot have been previously or

simultaneously invaded by other fungi, an occurrence that is rare.

In the case of the Turkey-X disease, the peanuts that were

responsible for the aflatoxin poisoning were from South America,

where the process used to harvest and dry the peanuts was

responsible for providing an environment that allowed for growth of

A. flavus and aflatoxin. Aspergillus flavus does not normally

contaminate grains and other crops while they are still in the

field. It is only after the grains are harvested and stored does A.

flavus, as well as other so-called " storage fungi " that have a low

moisture requirement, can the grain be invaded. Although conditions

favorable for growth of the A. flavus and production of aflatoxin is

narrow, the fungus is common and widespread in nature. It can be

found growing on various decaying vegetation where it may heat up

the substrate to as high as 113-122°F as it consumes the material.

The amount of aflatoxin formed differs as to the substrate on which

it is growing. Although the mycelial mass may be the same in each

substrate, the aflatoxin produced would be far greater in peanuts

than in say soybeans, where relatively very little would be

produced. Other seeds of cereal crops, wheat, corn, barley, oats and

sorghum are also generally of low-aflatoxin-risk. Weather and

climate were also contributing factors. Finally, the amount of toxin

produced will vary with the isolate of A. flavus. That is different

sources of A. flavus will produce different amounts of aflatoxins.

Some isolates of A. flavus may not even form aflatoxin.

Strange as this may sound, in some cultures, fungi are encouraged to

grow on certain foods in order to give them the desired taste. For

example, the Bantu tribes in Africa prefer the sour flavor of partly

spoiled corn to that of fresh corn and fungus is purposely allowed

to grow on the corn for this reason (Christensen, 1975). However,

this may only be a coincident, but they also have a very high

incident of primary liver cancer.

How much aflatoxin is too much?

Christensen (1972), over a period of several years, examined 100

different samples of black pepper from all over the world. In

dilution cultures of these samples, the number of fungus colonies in

whole or ground black pepper averaged 52,000 per gram/black pepper

and the upper range was over half a million per gram. These colonies

were mostly of A. flavus, A. ochraceus and A. versicolor. All three

species are known to be aflatoxin producers. Some samples of ground

pepper were caked lightly with fungus mycelium when first opened in

the laboratory and with time, a number of these became solidly caked

with mycelium.

How heavily contaminated is 52,000 to 500,000 colonies of fungi, per

gram? Lets make a comparison for what is acceptable levels of fungal

colonies isolated in other food products at the time Christensen

published his results. Wheat, for example, that is intended for

milling into flour seldom contains no more than a few thousand

colonies of fungi per gram of grain. If barley has as many as 10,000

colonies of the same kind of fungi per gram as in black pepper, it

would be rejected for malting in beer making. If breakfast cereals

or bread were as contaminated as black peppers, they would have so

musty an odor and taste that they would be too revolting to eat.

Apparently, the natural spicy odor and flavor of black, as well as

white pepper are potent enough to conceal the taste and odor of

these fungi. This is also true with many other spices.

One sample of black pepper was even found to contain a rodent

dropping and a piece of stone about the same size as the pepper.

Most samples contained peppers partly eaten by insects and partly or

mostly decayed by fungi and bacteria. However, you should keep in

mind that at the time Christensen carried out his little study,

quality control was not as much of an issue as it is today. With the

quality standards that are being enforced, presently, food products

with the type of contaminations described above would not be allowed

on the market.

What about the processed food prepared with A. flavus? At least, in

the commercial strains that have been utilized to prepare food in

the United States, the strains that have been tested have not been

found to produce aflatoxins. However, where these products are

prepared in a household or village industry, and the fungus is just

carried from one batch to the next, wild strains capable of

producing aflatoxin may contaminate them. In an investigation, in

the Philippines, not a single sample was found that was free of

aflatoxin. In such communities probably everyone was suffering to

some extent from chronic aflatoxin poisoning.

Mycotoxins in Other Species of Aspergillus, Penicillium and Fusarium

Aspergillus ochraceus and ochratoxin

Aspergillus ochraceus is also a species complex, and consist of nine

species. These species are common in soil, decaying vegetation, and

in stored seeds and grains undergoing microbial deterioration.

However, this fungus is seldom isolated from more than a small

percentage of seeds or grains that are undergoing microbiological

deterioration in storage because it is evidently not a good

competitor, as is also the case with A. flavus. This is a general

rule, but at the University of Minnesota, A. ochraceus sometimes has

been isolated from 40% or more of surface-disinfected kernels of

corns from bins in which deterioration was in progress. It has also

been the major organism in some lots of whole black pepper. Also,

samples of macaroni and spaghetti were found to be heavily invaded

by this species.

Production of ochratoxin, by A. ochraceus, was first described in

South Africa by Theron, et al. (1966), where it was isolated along

with a number of other fungi. In experiments done with this isolate,

the LD50 (the single dose that will kill 50 percent of the

individual animals tested) of ochratoxin for rats is 22mg/kg (= 22

milligrams of the toxin per kilogram of body weight of the rat), but

a lesser amount will result in severe liver damage. A single dose of

12.5 mg/kg (=12.5 milligrams of the toxin per kilogram of body

weight of the rat) was administered to pregnant rats on the tenth

day of gestation, and of the 88 fetuses involved, 72, or 81.8% died

or were resorbed. Ducklings seem to be equally sensitive to

ochratoxin as they are to aflatoxin.

Another fungus, Penicillium viridicatum, can also produce

ochratoxin, and is relatively common in stored corn and is a more

common producer of ochratoxin than A. ochraceus.

Aspergillus versicolor and sterigmatocystin

This species is another storage fungus. However, it is never found

as the only fungus or as the predominating fungus in deteriorating

cereals. Normally, by the time a grain sample has become very moldy,

A. versicolor, along with other Aspergillus species and usually

other filamentous fungi and yeasts as well. Some of the black pepper

mentioned earlier, as being decayed by fungi, was very heavily

invaded by A. versicolor, but not by this fungus exclusively.

One rather interesting case concerning this species took place, on

germinating barley, in a malthouse, in Scotland. The growth of A.

versicolor was so luxuriant on the germinating barley and produced

so many spores, that the workers who turned the malts with shovels

could not see one another because of the spore-filled air. The

owners and managers of the malthouse hired a mycologist to determine

how the fungus was getting in. They had assumed that the

contamination must have been due to the incoming barley. What the

mycologist concluded, however, was that there was a lack of sanitary

conditions, in the malthouse, and that everything in and around that

malthouse must have been thoroughly and heavily contaminated by A.

versicolor spores (and probably a lot of other fungi as well). So,

when conditions were favorable, i.e. when the barley was brought in,

the growth of fungi grew to spectacular quantities. Can you imagine

working in such an environment and having to inhale those spores?

This species, under the right conditions, produces sterigmatocystin,

a toxic compound given the name because the fungus once was called

Sterigmatocystis. The toxin is known to cause lung and liver tumors

in laboratory animals and has been implicated as the cause of

disease in calves that have consumed feed heavily invaded by A.

versicolor. Experiments carried out in which the fungus were grown,

on feed that was fed to calves, produced symptoms of the disease in

the calves. However, tests were not done to detect the toxin in the

calves. The toxin has also been detected in moldy coffee beans in

Africa, but no evidence indicates that even if these beans were used

to brew coffee that the toxin would be in the drink.

Aspergillus fumigatus and fumagillin

This particular species is known to be an animal pathogen. Infection

occurs through inhalation of spores and affects the lungs. Infection

may also occur in eggs and the fetuses of cows. However, it also

produces a metabolic product that may be considered a toxin or an

antibiotic. This species differs from the others that we have

discussed in that it is said to be thermophilic, that is, it is

found in substrate where there are extremely high temperatures, up

to 122ºF (=50ºC). This species is usually found on material that is

in the advanced stages of decomposition in which the substrate

temperature has been significantly raised by microbial decomposition.

Under the proper conditions, A. fumigatus produces fumagillin. This

compound is used as an amoebicide, that is, as a means to rid the

body of amoebae that are human pathogens and has been used

effectively in honey bees as well. However, the correct dosage of

this compound is critical. A little bit more than you need to get

rid of the amoebae and you will be getting rid of the patient as

well.

The Genus Fusarium

Species of Fusarium are widespread in nature as saprobes in decaying

vegetation and as parasites on all parts of plants. Many cause

diseases of economically important plants. For this reason, there

has been a great deal of research carried out in this genus by both

plant pathologist and mycologist. However, there are a number of

species that produce mycotoxins, mostly trichothecenes and

zearalenone. We will discuss a few common examples.

Fusarium tricinctum

The effects of the first trichothecene toxin, T-2, documented was in

the 1940s where it was associated with an outbreak of alimentary

toxic aleukia (ATA). At its peak, in 1944, the population in the

Orenbury District and other districts of the then USSR suffered

enormous casualties, more than 10 percent of the population was

affected and many fatalities occurred. The term alimentary toxic

refers to the toxin being consumed in foods and aleukia refers to

the reduced number of leucocytes or white blood cells in the

affected person. Other symptoms included bleeding from nose and

throat, multiple, subcutaneous hemorrhages.

The infected food in this case was millet, which made up a great

part of the diet of the people in the region, and at times, during

WWII, it was not uncommon to allow the millet to be left standing in

the fields over winter because bad weather in the fall prevented its

harvest at the proper time. During the late winter and early spring

the millet would become infected with a variety of fungi, including

F. tricinctum, and when the people gathered and ate this fungus,

many came down with what was diagnosed as ATA. Thousands were

affected, and many died. Locally, Joffe, a plant pathologist

determined the outbreak of ATA was caused by consumption of a toxin,

present in the millet, which had been contaminated by F. tricinctum.

This was a remarkable conclusion since this was 20 years before

aflatoxin was discovered. However, Joffe did not isolate or identify

the toxin involved and as a result his work remained unknown until

about 1965 when he presented a summary of his research at a

symposium on mycotoxins. The mycotoxin involved was later given the

common name T-2, and classified as one of several trichothecenes.

Fed orally to rats, it has an LD50 of 3.8mg/kg, which is lower than

that of aflatoxin, but still toxic enough.

Fusarium graminearum

Corn is a stable in many countries and is used as a major ingredient

in preparation of food for pigs and other domestic animals. Like

many other grains, the kernels can be infected with fungi before and

after harvest, and can affect the nutritional value of corn as food

or feed.

If the weather is rainy and the ears of corn are maturing in late

summer and early fall, F. graminearum may infect only a few to a

third of the kernels. Whatever amount of the ear is infected, all

the kernels in that portion becomes heavily infected and decayed by

the fungus. This fungus-infected corn is unattractive to pigs, as

well as other animals, and they refuse to it. For this reason, this

phenomenon has been called a refusal factor. Regardless of what the

composition of the rest of the feed, if it contains more than 5

percent of kernels with this refusal factor, the pigs will not eat

it and weight loss will occur. They will starve rather than consume

it. The infected corn contains an emetic compound produced by the

fungus, and if this corn is consumed by pigs, they suffer prolonged

vomiting, after which they sensibly refuse to eat more of the corn

(who said pigs were stupid?). The toxin involved is deoxynivalenol

(DON), also known as vomitoxin. The isolation and identification of

this toxin has occurred only within the last 25 years.

This is a serious problem if you look at it through the eyes of the

farmer. If you are a farmer and you have 800-1000 pigs and several

tons of feed mixed with corn, contaminated with vomitoxin, was

delivered to the farmer's feed bin on a Friday, and it is later

determined that the pigs will not eat it, then the farmer has a

serious problem. What are the pigs going to eat between Friday and

Monday?

Various methods have been tried to make the vomitoxin contaminated

corn more acceptable to pigs. Among some of the means that have been

tried are adding molasses to the feed to conceal whatever flavor or

odor makes it unacceptable to the pigs, heating the feed, in hopes

of destroying or inactivating whatever it is that is making the pig

refuse to eat it, and composting it so that the heat will break down

the toxin. However, none of these treatments have made the corn

acceptable to pigs and are impractical, anyway.

The detection of infected corn or feed is also a problem. Since we

are talking about mycotoxin here, the inability to isolate the

causal agent, F. graminearum, is not evidence that the mycotoxin is

absent. Long after a fungus has died off, mycotoxin secreted into

the substrate, will still be present. The refusal of pigs to eat

feed or corn is an indication that the refusal factor is present,

but not necessarily conclusive. There are a number of reasons as to

why pigs will refuse to eat. Pigs may be traumatized by being moved

to a new pen, strange surroundings or even being offered different

food. The only way that the toxin can be detected is to isolate,

purify and identify it by spectrographic or other analysis.

Use of Trichothecenes as a Biological Weapon

Yellow Rain

During the mid 1970s, when Vietnam was invading Laos, there were

stories of " yellow rain " in areas where entire villages were killed.

One eye witness account of such an event was told by a Hmong

refugee, in Thailand. While tending his poppies, outside of his

village, he and his family witnessed the bombing of their village by

the Vietnamese, in MIGS, with a yellow powder that came down like

yellow rain. Returning to the village, he found all of the animals

and most of the people were dead. The bodies were bleeding from the

nose and ears and their skin were blistered and yellowed. The few

people left alive, when he arrived, were " jerking like fish when you

take them out of the water " . These people also eventually died. The

witness took his family away from the village, but as they left they

felt shortness of breath and sick to their stomach. This story is

similar to other stories that were heard concerning yellow rain.

It was believed by the United States at that time that the Soviet

Union was somehow involved in what occurred in the Hmong village,

and medical teams were sent to investigate. However, because of the

remoteness of these villages, news of such attacks normally took 4

to 6 weeks to reach someone who could notify the medical teams. By

the time investigators reached a village, there was no evidence as

to what happened. It would not be until 1980 that a Defense

Department chemist recognized the symptoms described by victims of

the bombing as similar to trichothecene mycotoxicosis. Samples from

victims and from vegetation in the areas were tested and some were

found to contain trichothecenes. With this information, President

Reagan accused the Soviet Union of violating the Geneva

Convention and Biological Weapons Convention, which of course they

denied. However, these accusations would continue for three more

years.

While the accusations and denials were aired, the media and

scientific community gave a more critical examination of the yellow

rain story. The analysis that demonstrated Trichothecenes were being

used was initially based on a single leaf, collected where one of

the chemical attacks occurred. Subsequent specimens were collected

later that also showed Trichothecenes were present, but the ratio of

trichothecenes differed where it was found and was entirely absent

in some samples. In addition, little fanfare was given to the over

one hundred samples analyzed by the United States Army, which did

not find any indication of trichothecenes. The eye witness accounts

also came into question. Although it was implied that many villages

were attacked with yellow rain, all of the witnesses were from a

single refugee camp in Thailand, and even these accounts were

thought to be unreliable. For example in relating a story of the

bombing, one villager had initially said that 213 villagers were

killed, but in a later retelling, there were only thirteen people

killed and then forty. Further erosion of the government's yellow

rain story came about when a Yale University entomologist, whose

expertise was in Southeast Asian bees, examined yellow rain samples

and observed that they contained pollen from the native plants in

the area. Based on the appearance of these samples, it was concluded

that they were feces of bees. In one species of bees, present in the

area, there is a tendency for the bees to swarm when they defecated,

as a cleansing ritual, which could give the appearance of yellow

rain falling. News of such chemical attacks soon stopped and many

civilian scientists were convinced that the entire yellow rain

incident was a hoax that was carried out by the military to increase

funding for defensive chemical and biological weapons.

While a plausible alternative was given as to the cause of the

yellow rain, the eye witness accounts while questionable,

contradicted this theory. To date, the question as to what caused

the yellow rain has still not been satisfactorily resolved and may

never be.

Trichothecenes and the Lack of Population Increase in Europe

Something very interesting concerning mycotoxins in fungi has

recently come to light. Historical demographers, that is people that

study populations, their distribution, density and other such vital

statistics, have shown that long life and good health are a recent

phenomenon. Before 1750, in England for example, the life expectancy

of a member of the British peerage, that is one who has borne of

noble birth, was only 36.7 years, a hundred years later it had risen

to 58.4 years. Conditions were worse, and improvement slower among

the common folks. However, between 1750 and 1850, the population of

Europe almost doubled.

Before 1750 good health was a privilege of wealth, and not even then

did all the rich enjoy it. A commoner was often underweight,

stunted, sickly and occasionally deranged. They could not even

imagine the feeling of well being that we have today. There was a

constant battle with death, with results generally coming out in a

draw. There were great fluctuations in populations because of

mortality crises. After a mortality crisis, more people in a

community would marry and they would marry younger and would

eventually give rise to more children; so that if things

were " normal " , a community would tend to produce more babies than

corpses. But then pretty soon another crisis would come, e.g.,

disease, famine, natural disasters, etc., and the gain in population

would be wiped out.

The way in which populations were generally explained was in

Malthusian terms. That is populations were self regulating;

increases and decreases in birth rates were due to the availability

of resources, i.e. food, so if there was a lot of available food

there would be less death and more people would live to reproduce to

increase the population while if there was not enough available

food, there would be more deaths and fewer people would be around to

reproduce and the population would decrease. This is very sensible,

but in recent years it has been demonstrated, statistically, that

this is usually not the case.

What has been suggested, more recently, was that it was an increase

in fertility that was responsible for an increase in population, and

a decline in mortality, between 1750-1850. There have been several

reasons that have been proposed for the increase in fertility. One

reason that I became interested was the idea that changes in the

food supply was responsible for the increase in fertility. In a

recent book, by Matossian, Poisons of the Past, she puts forth

the theory that it was the change in diet of Europe that was

responsible for the tremendous increase in population.

During the 18th century, French adult peasants ate two to three

pounds of dark bread a day, when they could afford it. The rich and

affluent, which was less than 5 percent of the population, preferred

white bread. In the Mediterranean Basin, the diet of the poor

consisted of barley, buckwheat, wheat and after the 16th century

also included corn. North of the Alps and Pyrenees the poor made

their bread from rye or a mixture of rye and other grains, such as

barley, oats and buckwheat.

As you should now know cereal stables such as these come from plants

that are seldom free of molds, and it is Matossian's theory that it

was the consumption of such contaminated grains that had damaged the

immune system of the population of Europe that had relied on grain

as their staple and was probably largely responsible for a short

life span. T-2 and related trichothecenes are known to compromise an

individual's immune system. It would be a change of diet that would

begin to give individuals a longer life span. In addition, the

change in diet, which included potatoes also affected the birth

rate For example, recall that there was an increase in the Irish

population between 1750 and 1850. The reduced fertility in European

was believed to be due to the consumption of Rye that had been

contaminated with Ergot of Rye, a disease of Rye. This will be

another one of our topics on mycotoxins. The change to a diet which

consisted almost solely of potatoes rather than grains was

responsible for the increase in population. Matossian presented a

number of case studies to demonstrate that this had occurred, but

unfortunately, we don't have time to cover all of these cases.

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Most of this lecture was based on the following books. If you are

interested in reading a more detailed account of the above stories,

you may find them in Hamilton Library.

Christensen, C.M. 1975. Molds, Mushrooms, and Mycotoxins. University

of Minnesota Press, Minneapolis. 264 pp.

Hudler, G. 1998. Magical and Mischievous Molds. Princeton University

Press. 248 pp.

Hunter, B.T., 1989. Mycotoxins: A Significant Public Health Problem.

Consumer's Research 72 (6): 8-9.

Kendrick, B. 1992. Mycotoxins in Food and Feed in the Fifth Kingdom,

2nd ed. pp. 316-331. Mycologue Publications. Waterloo Ontario.

Matossian, M.K. 1989. Poisons of the Past: Molds, Epidemics and

History. Yale University Press, New Haven.

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Mycological Terms

Aflatoxins: First mycotoxins discovered, in 1960, produced by

Aspergillus flavus.

Aleukia Toxic Aleukia (ATA): Condition associated with consumption

of trichothecenes mycotoxin, T-2, produced by Fusarium tricinctum.

Some symptoms of condition include low white blood cell count,

multiple subcutaneous hemorrhages and bleeding from nose and throat.

Aspergillus flavus: Species complex in which first mycotoxins,

aflatoxins, were discovered.

Aspergillus fumigatus: Species of fungi producing

mycotoxin/antibiotic fumagillin.

Fumagillin: Compound produced by Aspergillus fumigatus, an example

of the fine line between something that is medicinal and a

poisonous. Although classified as a mycotoxin, it is also

effectively used as an antibiotic for amoebic parasites, in human as

well as honeybees. However, dosage is very important here, if too

much is used, it can be fatal.

Fusarium graminearum: Species associated with production of

mycotoxin, vomitoxin, which causes pigs and other animals not to

consume food when present. Pigs would initially eat food, containing

toxin, but after a prolonged period of vomiting, refused to consume

more food with toxin. Refusal to eat leads to weigh loss in animals.

Fusarium tricinctum: Species producing trichothecenes associated

with alimentary toxic aleukia (ATA). First documented in 1940s, of

USSR.

Kwashiorkor: Protein deficiency disorder of children in various

overpopulated countries in the world.

Matossian: Historian and author of Poisons of the Past.

Believed that the population depression that occurred in Europe

before 1750 was due to consumption of moldy grains that contained

mycotoxins. It was believed that such mycotoxins reduced fertility

and life span of individuals, and that it was not until a change in

diet from grains to potato did the situation improved. Between 1750

and 1850, population of Europe doubled as a result of change in

diet.

Mycotoxicoses: The effects of poisoning from mycotoxins.

Mycotoxins: Literally poisons from fungi, Myco = fungus, toxins =

poison.

T-2: Trichothecene mycotoxin produced by Fusarium tricinctum that is

associated with ATA.

Vomitoxin: Name given to trichothecenes that causes pigs and other

animals to refuse food containing this mycotoxin. Pig would

initially eat food with toxin, but after a prolonged period of

vomiting, the pig would refuse to eat it even when hungry. It caused

weigh loss because of refusal to eat.

Yellow Rain: Name given to yellow, powdery material said to have

been dropped from MIGs, during attack of Hmong villages, in Laos, by

Vietnamese, in mid 1970s. Powdery material was said to be a

trichothecenes that quickly killed villagers.

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Questions to Think About

What was the inherent difficulty of demonstrating that a metabolic

product produced by fungi was the cause of illnesses?

When an organism produces a chemical, it is usually for a reason.

What reason can you give for fungi producing mycotoxins.

Although Aspergillus flavus is known to produce aflatoxins, it is

also utilized in the making of a number of food products, miso, sake

and soy sauce, to name a few. Why is it that the makers of these

food products are not concerned about aflatoxin?

Although the existence of mycotoxins was suspected before its actual

discovery, and there were several cases in which the fungal

contaminated food material was known to be the source of poison, for

example Joffe's work in 1940. Yet, it was not until 1960, when

research was being carried out on the Turkey-X disease that the

existence of mycotoxin was accepted. What was different about this

research that finally made everyone accept that it was a poison

produced by the fungus that was causing illnesses to occur rather

than just the consumption of the fungus infected food?

Aspergillus fumigatus produces fumagillin, which can either be

classified an antibiotic or mycotoxin. Can you explain how a single

compound can be classified as both?

What is your opinion on the " yellow rain " being used as a biological

weapon in Laos, during the mid 1970s?

Briefly summarize the reason given by Potossian on the lack of

population growth, in Europe, until the late 18th. Century.