©2001 CRC Press LLC

chapter ten

Mycotoxins and other toxins
from unicellular organisms

Anything green, that grew out of the mould,
Was a wonderful drug to our fathers of old.

— Anonymous

Introduction

Mycotoxins are a group of chemically diverse and complex substances
present in a wide variety of filamentous fungi (molds). They are secondary
metabolites of fungal metabolism of uncertain function. Some may have a
survival advantage by virtue of their toxicity to competing organisms in
the microenvironment. The biological function of others is unclear, but
many have significant biological activity and several are toxic to mammals.
They therefore have significant public health and economic implications.
It is estimated that 25% of the world's annual food crops are contaminated
by mycotoxins.
Some mycotoxins are of interest to pharmacologists and toxicologists
because they have served as research tools to study cell function and to
identify various types of neurotransmitters and blocking agents. Poisonous
mushrooms are solid (not filamentous), spore-forming fungi that also con-
stitute a health hazard and which have historic, pharmacological signifi-
cance, but they are considered separately in Chapter 11.

Some human health problems due to mycotoxins

experiencing visions and hallucinations. These frequently involved attacks
by Satan in various guises (wild beasts, soldiers, and women) that either
physically attacked him or tempted him. Contemporary witnesses claimed
that he behaved like an individual being physically abused. Because the
signs and symptoms of ergot poisoning resembled his attacks, sufferers in
the Middle Ages attached religious significance to them and they came to
be known as “St. Anthony's Fire” (or sometimes “Holy Fire”). In 1100, the
Monastery of the Hospitillers of St. Anthony was established at La Motte in
France. It became the site of pilgrimages by those afflicted with this malady.
The signs and symptoms included intense burning pain in the extremities
followed by a blackened, necrotic appearance; hence the association with
fire. Epidemics of madness in the Middle Ages may also have been the result
of ergotism.
Ergot was employed as an abortifacient long before it was known to be
the cause of St. Anthony's Fire, and this use continued well into the nine-
teenth century when it was finally abandoned because of its highly toxic
nature. One of the components of ergot is still employed to stop

postpartum

hemorrhage because it constricts blood vessels and contracts the uterus. To
summarize, the symptoms of ergotism include drowsiness, nausea, vomit-
ing, muscle twitch, staggering, gangrene, hallucinations, and abortion. Ergot
contains a veritable potpourri of pharmacological agents that were charac-
terized by Sir Henry Dale in the early 1900s and contributed to the devel-
opment of many new drugs.
The active components of ergot, the ergot alkaloids, are all derivatives
of lysergic acid. They bear some molecular resemblance to adrenaline (epi-
nephrine), dopamine, and serotonin — all central neurotransmitters. Meth-
ysergide is the precursor of LSD. Two derivatives still have medicinal appli-


and

A. parasiticus

. These molds grow
abundantly on many kinds of plants in very hot conditions. Peanuts are
especially prone to infection. Taste is not affected and the toxins are heat
stable. Aflatoxin B

1

(AFB

1

) is the most frequently encountered member of
the group and it is a potent carcinogen in experimental animals (rodents,
birds, and fish). Rats fed a diet containing 15 ppb AFB

1

develop hepatitis,
which is often followed by cancer of the liver. Epidemiological studies of
populations in Uganda, Kenya, and Thailand have shown a close correlation
between the incidence of liver cancer and the consumption of food contain-
ing aflatoxins. It is estimated that the risk factor for liver cancer is increased
10 times by AFB

1

P450 (Cyp) 3A4 is largely responsible for the formation from AFB

1

of the
8,9-

endo

-epoxide, a genotoxic metabolite. Cyp 1A2 also forms this epoxide
but is much less active. It forms other, nongenotoxic metabolites and thus
may play a role in detoxification. An inhibitor of Cyp 1A2, alpha-naphthofla-
vone, increased the formation of the 8,9-epoxide. The

exo

-epoxide of AFB

1

(i.e., preformed before ingestion) is detoxified by conjugation with glu-
tathione. Experimental evidence suggests that species differences in toxicity
are due to differences in the rate and extent of conjugation with glutathione.

©2001 CRC Press LLC

Whereas rat liver cytosol conjugated endo-epoxide, mouse liver cytosol con-
jugated the

exo


. Livestock (mammals) may also be affected. Death
has occurred in humans consuming an estimated 6 mg/day of aflatoxin B

1

.
In North America, there is concern about the long-term effects of con-
suming low levels of aflatoxins and monitoring systems are in place. Fortu-
nately, the mold is not adapted to the colder northern climate so that Cana-
dian-grown peanuts are free of the toxins. The majority of peanuts and
peanut butter are still imported from subtropical climes, however. Peanuts
are not the only source of these toxins. Because of drought conditions in the
United States in the summer of 1988, up to 30% of the corn crop may have
been contaminated. The Quaker Oats Co. was turning away almost one
truckload in five at its Cedar Rapids (Iowa) plant in the fall of 1988. The
company tests six samples from each truck. Inspections of the 340,000 tons
of corn crossing the Canada–U.S. border annually were stepped up in
1988–1989 by Agriculture Canada. The general structure of aflatoxins is
shown in Figure 37.

Fumonisins

Produced by

Fusarium monilforme

and

F. proliferatum

around the world. Fumonisins are toxic for many species of animals, espe-
cially horses, and outbreaks have caused numerous deaths in horses and

©2001 CRC Press LLC

swine in Texas, Iowa, and Arizona. Severely infected corncobs may contain
up to 900 mg/kg. Symptomatology in various species is as folllows:
•

Swine

: vomiting, convulsions, sudden death, abortion, pulmonary
edema (porcine pulmonary edema syndrome). Symptoms may
occur at levels above 20 to 50 mg/kg of FB

1

.
•

Poultry

: ataxia, paralysis, sudden death, stunted growth. Toxicosis
occurs at levels of contamination of 10 to 25 mg/kg of FB

1

.
•


de novo

sphingolipid synthesis and sphingolipid turnover. FB

1

has been
shown to inhibit sphingosine (sphinganine) N-acetyltransferase, leading to
the accumulation of sphyngoid bases. This has been shown to stimulate DNA
synthesis, and it is hypothesized that this interference with normal cell
function could account for the toxicity of fumonisins.
The fumonisins are themselves analogs of sphingosine, with structural
similarities to the phorbol esters, which are known carcinogens (see
Chapter 11). The general structure of fumonisins is shown in Figure 37.

Figure 37

Chemical structures of aflatoxins and fumonisins.
O
O
O
O
AFB
1
R1 R3
R2R1
CH3
NH2
CH3CH3
OH


Penicillium

species. It
has been shown to be embryotoxic and teratogenic in several laboratory
species of mammals (pig, dog, mouse, rat) and birds and is therefore viewed
as a potential human teratogen. Acute effects in animals (including swine
and poultry) include renal and hepatic destruction. Both humoral and cel-
lular immune systems are adversely affected. Contamination of bread and
cereals has been documented in parts of central Europe (Yugoslavia, Bul-
garia, Poland, and Germany) and levels have been detected in human milk,
urine, blood, and kidneys.
Patulin is potentially a carcinogenic toxin produced by several species
of fungi, including some

Penicillium

spp. It is a highly potent inhibitor of
RNA polymerase, having a strong affinity for sulfhydryl groups. It therefore
inhibits many enzymes. Teratogenicity has not been demonstrated in mam-
mals but embryolethality occurs at higher doses (2 mg/kg i.p.). A common
source of patulin is

Penicillium expansum

, a common spoilage microorganism
in apples. Apple juice can sometimes contain significant amounts of patulin.
Acute toxicity in rodents is manifested primarily as gastrointestinal symp-
toms, including hemorrhage. Carcinogenicity studies in rats were negative,
but clastogenic activity has been shown in some systems.

©2001 CRC Press LLC

common offenders, especially in eastern Canada. They may produce a host
of toxins with potent pharmacological actions.

Fusarium life cycle

Fusarium graminearum

is the fungus responsible for maize ear rot in corn and
head blight in wheat. Its life cycle is typical of all

Fusaria

. Spores survive in
crop debris from the previous season (stubble, stalks and seeds) to re-infect
the next year's crop. Intensive farming practices that involve planting suscep-
tible species in the same fields year after year thus favor the spread of infec-
tion. Birds such as starlings and red-winged blackbirds, which puncture the
corn kernels to eat the milk, may also spread spores. Insects such as the picnic
or corn-sap beetle seek out damaged kernels and also may spread spores.
Certain weather conditions favor the spread of infection. Fungus growth
is favored by warmth (15 to 35°C) and by surface wetness for more than
48 hr. After the infection is established, weather is not critical to the produc-
tion of the toxin. Mold growth will continue throughout the season, and
even afterward if not properly dried or if storage conditions are poor (too
damp, too warm, poor air circulation). Mold growth can even occur during
feed preparation and in poorly cleaned feed troughs. Late harvest may allow
the growth of another fungus,



tolerant. In one study, laying hens actually preferred a diet containing 5 ppm
DON in preference to clean feed. These differences are due, in part, to
differences in absorption and, in part, to differences in biotransformation
and elimination. Studies with radiolabeled DON indicated that sheep
absorbed 9% or less of an orally administered dose, in turkeys 20% or less
was absorbed, whereas pigs absorbed up to 85% of a single oral dose.
Intravenous administration of radiolabeled DON in sheep revealed an
initial distribution phase (t

1/2

= 18 min) followed by an elimination phase
(t

1/2

= 66 min). A glucuronide conjugate was formed and comprised 15 to
20% of plasma levels. In turkeys there was an extremely rapid distribution
phase (t

1/2

= 3.6 min), a rapid elimination phase (t

1/2

= 46 min), and the
formation of a conjugate (probably glucuronide) comprising up to 10% of
the total dose. Again, swine showed a much different picture. There was a

to tissues and then slowly released back into the plasma, yielding the slow,
terminal elimination phase. Turkeys also had a very large Vd (2.33 L/kg), but
they also had an extremely rapid clearance (35.0 mL/min/kg), indicating that
DON was rapidly distributed to tissue compartments but not held there.
Thus, the extreme sensitivity of swine to DON is the result of:
• High oral bioavailability
• Wide distribution to tissues
• Slow elimination from the body
• Minimal detoxification through biotransformation

©2001 CRC Press LLC

Other trichothecenes

T-2 and HT-2 toxins and diacetoxyscirpenol are more toxic than DON and
cause reduced feed intake, vomiting, irritation of the skin and gastrointesti-
nal tract, neurotoxicity, teratogenicity, impaired immune function, and hem-
orrhage. Adverse effects seen in farm animals are generally caused by mix-
tures of these toxins rather than by a single toxin. Blending of several grains
in the preparation of feed may further contribute to the toxic diversity of
the mixture. Potentiation of effects may occur. Thus, DON at the subthresh-
old level of 1 mg/kg plus low (ppb) concentrations of T-2 and other uniden-
tified toxins may cause severe toxic manifestations in a sensitive species such
as swine. The chemical structures of some of these toxins are shown in
Figure 38.

Figure 38

Chemical structures of several mycotoxins.
HO

O
O
H
HH
Zearalenone
O
O
Deoxynivalenol ( DON, vomitoxin )
T - 2 : CH
3
COO at R
1
and R
2
, ( CH
3
)
2
CHCH
2
COO at R
3
HT - 2 : OH at R
1
, CH
3
COO at R
2
, (CH
3

In corn, the tips of cobs may have shriveled, highly infected kernels con-
taining up to 3000 mg/kg of DON. Grain dust may be very contaminated.
Screening and blowing will remove much of the dust, particles, and with-
ered kernels.
Wet milling of corn has been shown to remove about two thirds of the
T-2 toxin, but milling had little effect on the DON content of flour from hard
wheat, nor did baking the flour into bread. Some milling procedures may
actually increase the DON content of the finished product. In mild infections,
washing and roasting may significantly reduce toxin levels.

Chemical treatments

Laboratory tests have shown that moist ozone, ammonia, microwaving, and
convection heating reduce DON concentrations in moldy grain. Aqueous
sodium bisulfite plus heat effected a complete detoxification. Studies have
shown that this technique resulted in normal feed intake and weight gains
when contaminated corn was treated and fed to swine.

Binding agents

The addition of binding agents, such as bentonite, anionic and cationic resins,
and vermiculite-hydrobiotite were tested on the toxicity of T-2 in rats. Ben-
tonite prevented T-2 toxicosis by blocking intestinal absorption. Polyvi-
nylpyrrolidone or ammonium carbonate had no effect on DON toxicity in
swine. Alfalfa fiber has been shown to partially overcome the growth-
depressing effect of zearalenone in rats but not the estrogenic effects in swine.

Other techniques

Dilution of contaminated feed with clean feed will improve palatability and

is 390 min for the cat and 300 min for humans.
The question of mycotoxin residues in human food sources remains
largely unanswered, but studies indicate that DON residues are not a prob-
lem. Feeding very high levels of DON to dairy cattle resulted in only trace
quantities in milk; and when fed to poultry, no appreciable tissue residues
were measured. Again, this is the consequence of the species pharmacoki-
netic characteristics.

Other toxins in unicellular members of the plant kingdom

Many soil organisms produce substances that are toxic to others and a
continual state of chemical warfare exists in the microenvironment. These
organisms provide the source of all of our antibacterial and antifungal anti-
biotics, many of which have significant mammalian toxicity. Many others
were tested and discarded because of their high toxicity. Some antibiotics
are teratogenic and are used in the treatment of cancer because of their effects
on cell reproduction (e.g., actinomycin D, doxorubicin, adriamycin). Some
organisms may be directly responsible for poisonings in humans.
The blue-green algae called

Cyanobacteria

produce pentapeptides that
are hepatotoxic and have caused numerous deaths when they contaminate
drinking water. Some strains of that ubiquitous organism

Staphylococcus
aureus

are capable of producing a protein enterotoxin, 1


Further reading

Eaton, L. and Gallagher, E.P., Mechanisms of aflatoxin carcinogenesis,

Annu. Rev.
Pharmacol. Toxicol

., 34, 135–172, 1994.
Marasas, W.F., Fumonisins: their implications for human and animal health,

Nat.
Toxins

, 3, 193–198, 1995.
Nair, M.G., Fumonisins and human health,

Ann. Trop. Paediatr

., 18 (Suppl.),
S47–S52, 1998.
Pitt, J.I., Toxigenic fungi and mycotoxins,

Br. Med. Bull

., 56, 184–192, 2000.
Rheeder, J.P., Marasas, W.F.O. et al.,

Fusarium moniliforme



1

.
A molecular mechanism for carcinogenesis associated with

Fusarium monili-
forme

,

J. Biol. Chem

., 269, 3475–3481, 1994.
Shepphard, G.S., Theil, P.G., Stockenstrom, S., and Sydenham, E.W., World-wide
survey of fumonisin contamination of corn and corn-based products,

J. AOAC
Int

., 79, 671–687, 1996.
Shier, W.T., Sphingosine analogs: an emerging new class of toxins that includes the
fumonisins,

J. Toxicol

., 11, 241–257, 1992.
Ueng, Y.F., Shimada, T., Yamazaki, H., and Guengerich, F.P., Oxidation of aflatoxin
B1 by bacterial recombinant human cytochrome P450 enzymes,


c. DON causes vomiting and poor weight gain in swine.
d. Other trichothecenes are less toxic than DON.
2. Which of the following statements is/are true?
a. All varieties of wheat and corn are susceptible to fungal infections
in varying degrees.
b. Rapid drying of grain minimizes the risk of post harvest mould
growth.
c. Mycotoxin toxicity in livestock usually results from mixed fungal
contamination.
d. There is no practical way of detoxifying corn and wheat contam-
inated with mycotoxins.
3. Which of the following statements is/are true?
a. There is no evidence that fumonisins are carcinogenic for humans.
b. Zearalenone has estrogen-like activity.
c. Aflatoxins are not toxic for poultry.
d. Ochratoxin causes renal damage in swine and poultry.
4. The symptoms of ergot poisoning:
a. Are similar for humans and animals.
b. Include nausea, dizziness, burning pain in the extremities, and
abortion.
c. Arise primarily from consuming rye contaminated with ergot
alkaloids.
d. Are caused by ergot alkaloids produced by

Fusarium moniliforme

.
5. Which of the following statements is/are true regarding fumonisin
B



8. Regarding aflatoxins:
a. The mold that produces them is very hardy and survives in cold
climates.
b. They are oxygenated heterocyclic compounds.
c. They cause hepatic damage but are not carcinogenic.
d. Epidemiological studies have shown a high degree of correlation
between liver cancer and the consumption of foods contaminated
with aflatoxin B

1

.
9. Regarding detoxification of grains contaminated with mycotoxins:
a. A technique that works on one mycotoxin may not work on
others.
b. DON concentration may be reduced by heating.
c. Binding agents may reduce the toxicity of T-2.
d. Moist ozone and ammonia have reduced DON concentrations in
lab tests.
10. Regarding DON toxicity:
a. Swine are more susceptible than poultry or ruminants.
b. Turkeys form a conjugate and rapidly eliminate DON.
c. Sheep form a glucuronide conjugate.
d. A high volume of distribution (Vd) is generally associated with
higher toxicity in that species.
11. Match the statement with the correct toxin or effect.
a. Cyanobacteria.
b. Blocks acetylcholine release from peripheral nerve endings.
c. Adriamycin.

ing AFB

1

epoxide.
d. The 8,9-epoxide is formed exclusively by Cyp 1A2.
e. Cyp 3A4 is the main source of 8,9-

endo

-epoxide.
f. Species differences in the rate and extent of glutathione conguga-
tion of 8,9-epoxide may account for differences in toxicity.

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Answers

1. B
2. A
3. C
4. A
5. A
6. E
7. B
8. C
9. E
10. A
11. i = d, ii = a, iii = c, iv = b
12. a. True