Mycotoxins: of Molds and Maladies

[Guest guest]

Top image credit: Aspergillus Photo:A RS/USDA

http://www.ehponline.org/docs/2000/108-1/focus.html

Mycotoxins: of Molds and Maladies

From the beginning of organized crop production through present-day

agriculture, mycotoxins--toxic metabolites produced by fungi--have

presented health risks to both human and animal populations.

Mycotoxins are secondary metabolites produced by certain molds that

infect food crops in the field and during storage. Depending on the

quantities produced and consumed, mycotoxins can cause acute or

chronic toxicity in the animals and humans who eat contaminated

crops or foods prepared from them. Health effects of mycotoxins may

include immunological effects, organ-specific toxicity, cancer, and,

in some cases, death. Agricultural workers are also at risk for

dermal and respiratory exposures during crop harvest and storage.

Mycotoxin contamination is a worldwide problem affecting staple

crops such as corn (maize) and small grains (such as wheat), as well

as tree nuts, peanuts, sorghum, and many others. Many countries

regulate the maximum allowable concentrations of specific mycotoxins

in food commodities and animal feed. Until recently, dietary and

occupational exposures were the primary areas of concern, but with

growing attention being paid to the problems associated with indoor

molds and respiratory exposures, researchers are recognizing that

the potential scope of mycotoxin exposures is broader than

originally suspected. This scope now includes inner-city

environments, with a special focus on older or poorly maintained

structures that are more susceptible to harboring molds.

Researchers are also recognizing that specific groups within a

population may be more vulnerable to exposure than the population at

large. For example, in the United States, the Hispanic community

consumes a greater quantity of corn products compared to the general

population. Because corn is vulnerable to contamination by several

mycotoxins, it is possible that individuals within the Hispanic

community are exposed to higher dietary levels of mycotoxins than

the average American. This higher exposure could place them at a

greater risk of health problems.

The effects of mycotoxins have been recorded for millennia in

sources as diverse as 5,000-year-old Chinese texts, the Old

Testament of the Bible, and numerous scientific journals. As

research into these compounds and their effects has accumulated over

the last several decades, mycotoxins have been revealed to be an

extremely diverse group of compounds.

During the Middles Ages, periodic epidemics of St. 's fire,

now known as ergotism, afflicted countless individuals who had

consumed moldy rye. Gangrenous ergotism, the form that inspired the

medieval name, was accompanied by fiery pain, swelling, and gangrene

in the limbs. Convulsive ergotism, a second form of the toxicosis,

was accompanied by convulsions and hallucinations, among other

symptoms. Both forms could be fatal. Research in the 1940s

identified ergot alkaloids produced by Claviceps spp. as the

mycotoxigenic source of ergotism. Ergotism has occurred very rarely

in recent decades; with regard to agricultural crops, current

research focuses on the toxigenic and carcinogenic potentials

associated with molds including species of Aspergillus and Fusarium.

With regard to indoor molds and respiratory health issues,

Stachybotrys chartarum (formerly S. atra) contributes a challenging

new facet to the field of mycotoxicology.

A Centerpiece of Mycotoxin Research

" Monolithic " is how D. Groopman, a professor of environmental

health sciences at the s Hopkins University School of Public

Health in Baltimore, land, describes the literature on

aflatoxins--mycotoxins produced by Aspergillus spp. " As far as the

epidemiology in people goes, the overabundance of literature covers

aflatoxins, " he says. " Aflatoxin levels are regulated not only by

the Food and Drug Administration [FDA] and the U.S. Department of

Agriculture [uSDA] but also in world commerce. I don't believe

that's true for almost any of the other mycotoxins. " This emphasis

reflects the central role of aflatoxins, especially aflatoxin B1

(AFB1), in mycotoxicological research since the early 1960s.

Primarily produced by A. flavus and A. parasiticus, AFB1 causes

liver and kidney toxicity in several species and is most prominently

known as a potent liver carcinogen in humans and animals.

Aspergillus spp. exist worldwide and live off of a number of crops,

although corn and peanuts are the most commonly contaminated

commodities. A. flavus and A. parasiticus are the most common forms

of Aspergillus and are also capable of producing other forms of

aflatoxin. Frequently, there is contamination with multiple forms of

aflatoxin. However, regardless of which aflatoxins are produced,

AFB1 is always among them, and it is the most toxic.

AFB1 has been shown to be mutagenic in many in vitro model systems

and is a proven carcinogen in many animal species, including rodents

and nonhuman primates. The primary cancer site in these studies has

been the liver, but in some cases a link has been demonstrated to

lung, kidney, and colon tumors. In human epidemiological studies,

chronic dietary AFB1 exposures have been strongly linked to

increased incidence of liver cancer. On the basis of this

information, AFB1 is classified as a known human carcinogen by the

International Agency for Research on Cancer and the National

Toxicology Program.

The carcinogenic potency of AFB1 is not manifested until it

undergoes activation by the cytochrome P450 and other oxidative

enzymes. These enzymes transform the aflatoxin into several

products, including the genotoxic AFB1-exo-8,9-epoxide. This epoxide

can be shunted by glutathione S-transferase in the liver, but some

will intercalate, or wedge itself, between DNA base pairs. In this

position, the epoxide rapidly reacts with the DNA to produce an N7-

guanyl adduct. As demonstrated in several animal models, these

adducts are produced in the greatest amounts in the liver, although

some are also produced in the kidney or lung. Through DNA repair and

chemical stability mechanisms, AFB1-DNA adducts can be removed and

excreted, but some adducts prove resistant to repair, thereby

setting the stage for mutation events and carcinogenesis. The

potential for AFB1-induced liver cancer is enhanced in individuals

who are also infected with the hepatitis B virus, a recognized

carcinogenic virus. In AFB1-exposed populations, examination of

liver tumors reveals a high incidence of a specific p53 tumor

suppressor gene mutation. In AFB1-exposed individuals infected with

the hepatitis B virus, this mutation is associated with 50-60% of

the tumors. By understanding the mechanism of action of AFB1, we are

identifying guideposts for developing intervention strategies, says

Groopman.

The Depth of the Field

Not all carcinogenic mycotoxins act through a genotoxic mechanism,

and the fumonisins provide a case in point. The fumonisins--B1, B2,

and B3--are produced by Fusarium spp. that grow on corn, most

importantly F. moniliforme. In horses, relatively low exposures to

fumonisins have been shown to cause equine leukoencephalomalacia, a

disorder characterized by brain hemorrhage and necrosis, followed by

death. Horses may also suffer liver damage and possibly a degree of

kidney damage following dietary exposure to fumonisins. Liver and

kidney effects are more pronounced in other species such as rodents,

sheep, and rabbits. In swine, high doses of exposure to fumonisins

seem to especially affect the lungs, leading to porcine pulmonary

edema, a fatal condition in which fluid collects in the lungs. Low

exposures result in reduced feed consumption. Early research in

animals showed that fumonisins are potent cancer promoters and

potentially weak initiators. The National Toxicology Program, along

with the FDA's National Center for Toxicological Research in

Jefferson, Arkansas, and Center for Food Safety and Applied

Nutrition in Washington, DC, released the results of a massive study

of the toxicity of fumonisin B1 in May 1999. Their data showed that

fumonisins are carcinogenic in rodents, although response differed

by species and sex: male rats fed fumonisin B1 developed liver and

kidney cancers, while female mice developed liver cancer.

With regard to human toxicity, epidemiological data from southern

Africa and China suggest a strong link between dietary fumonisin

exposure and esophageal cancer. Human epidemiological studies,

however, are not definitive, says Ken Voss, a research

pharmacologist at the USDA Toxicology and Mycotoxin Research Unit in

Athens, Georgia. " There are suggestive data that the fungus and the

fumonisins are associated with esophageal cancer, " he says. " But

there are enough confounding dietary and environmental factors that

the correlation, although tantalizing and suggestive, is as yet far

from being proven. " According to Voss, it has been shown that

fumonisins have measurable and repeatable toxic effects in animal

models.

Fumonisin toxicity seems to be mediated through inhibition of

ceramide synthase, a key enzyme in the sphingolipid biosynthetic

pathway. " To put it in layman's terms, " says Voss, " the entire

metabolism of sphingolipids in the cell is disrupted. " The potential

ramifications of this disruption can be far-reaching, he says. Until

15-20 years ago, sphingolipids were considered as having a purely

structural role in cells; however, sphingolipid molecules and their

derivatives are now recognized as very biologically active

compounds. These compounds, says Voss, either initiate or act as

messengers for many life-or-death decisions that the cell has to

make. Such decisions include whether the cell embarks on apoptosis

(cell death) or enters the cell cycle and replicates. From this

point, he says, there are a host of potential steps and

intermediaries leading to toxigenic or carcinogenic events.

Fusarium and Its Mycotoxins

In addition to fumonisins, Fusarium spp. produce several other

mycotoxins. F. graminearum and F. culmorum, molds that contaminate

corn, barley, wheat, and other crops, are capable of producing the

toxins zearalenone and deoxynivalenol (also called vomitoxin).

Different toxigenic species of Fusarium grow under different sets of

climatic conditions. " The production of these compounds depends on a

number of different conditions, " says Retha Newbold, a supervisory

research biologist at the NIEHS. " Just to have a product that is

contaminated with mold is not to assume that mycotoxin is present.

The mold may be there, but it may produce different levels of

mycotoxins, or even different mycotoxins, depending on . . .

different conditions. "

Although zearalenone has low acute toxicity, it exhibits marked

estrogenic effects in some species. Zearalenone and its metabolites,

particularly -zearalenol and ß-zearalenol, have been shown to bind

to estrogen receptors in experimental systems. Their estrogenic

potential seems to fall between that of the endocrine-disrupting

organochlorine pesticides and the more estrogenic compound

diethylstilbestrol. Newbold indicates that the estrogenicity of

zearalenone and its metabolites differs depending on the tissue and

the species. For example, swine are especially sensitive and

experience hyperestrogenism leading to reproductive problems and

infertility following dietary zearalenone exposures. Other species

such as cattle and sheep seem more resistant to zearalenone but may

still experience some incidence of infertility, decreased milk

production, and spontaneous abortion after ingesting high doses.

Still other species, particularly chickens, appear even less

sensitive.

Fusarium moniliforme

Photo: De Lucca

ARS/USDA

It has been demonstrated that zearalenone and its metabolites may

cause carcinogenesis or teratogenesis in some species, but further

research is needed. Further research is also needed with regard to

human toxicity. Currently, the International Agency for Research on

Cancer classifies zearalenone as a 2A carcinogen, the highest

possible classification when categorical human epidemiology is

absent. Several countries have already established maximum allowable

concentrations of zearalenone in food ranging from 0 to 1,000

micrograms per kilogram. Data on human toxicity are strongest with

regard to estrogenic effects. For example, zearalenone was

considered a possible etiological agent for precocious pubertal

changes that were observed among Puerto Rican children for several

years beginning in 1979. Thousands of children, some of whom were

shown to have zearalenone or its derivatives in their blood,

reportedly experienced symptoms. However, as other estrogens

(phytoestrogens or residues of animal growth promoters) were

potentially present in the children's diets, this outbreak might

have stronger implications with regard to zearalenone's contribution

to the total environmental estrogen burden. Recent investigations

using in vitro systems bolster the idea that zearalenone interacts

with human estrogen receptors. For example, Craig Dees, a scientist

in the Health Sciences Research Division of the Oak Ridge National

Laboratory in Tennessee, and colleagues reported that zearalenone

stimulates estrogen-receptor human breast cancer cells to enter the

cell cycle in vitro [EHP 105(suppl 3):633-636 (1997)].

" For people who are studying endocrine disruptors and who are

actually looking for some of the potential health effects during

development, this [mycotoxin] is one of their concerns, " says

Newbold. " But I certainly don't think it has received the attention

for human health that it should have in the United States. "

With regard to research, zearalenone seems to be overshadowed by

deoxynivalenol, a more demonstrably toxic Fusarium metabolite. This

mycotoxin has been linked to large-scale poisonings, human disease,

and animal production problems throughout the world. Deoxynivalenol

is one of the most common mycotoxins contaminating grains. It

belongs to a class of compounds called trichothecenes, to which

several other mycotoxins belong. Although deoxynivalenol is the

least toxic of the trichothecenes, its toxicity is still substantial

in both animals and humans. In large enough acute doses, it causes

nausea, vomiting, and diarrhea and destroys blood cells. Animals,

particularly pigs, demonstrate feed refusal and weight loss at lower

doses. Deoxynivalenol has also been shown to have immunological

effects in animal models. For example, research reviewed by J.

Pestka, a professor of food science and human nutrition at Michigan

State University in East Lansing, and colleagues in the May 1996

issue of the Journal of Toxicology and Environmental Health

demonstrates that deoxynivalenol interferes with normal immune

system functioning in mice. They concluded that deoxynivalenol

induces cytokines, immune system factors that help direct an

inflammatory response. In the model they reviewed, mice exposed to

deoxynivalenol developed symptoms similar to human IgA nephropathy,

a kidney disorder characterized by inflammation.

Expanding Frontiers in Mycotoxicology

Trichothecenes may also be at the root of an outbreak of idiopathic

pulmonary hemorrhage among infants in Cleveland, Ohio [EHP 107(suppl

3): 495-499 (1999)]. Among infants, pulmonary hemorrhage, or

episodes of bleeding in the lungs, can arise from several causes

such as injury or some forms of pneumonia. Unexplained, ongoing

episodes of bleeding, as seen with the infant patients in Cleveland,

is much rarer. Between 1993 and 1998, physicians at the Rainbow

Babies & Children's Hospital in Cleveland saw 37 cases of pulmonary

hemorrhage among infant patients; in the preceding 10 years, only

three such cases had been encountered. Nearly all of the 37 infants

were brought to the hospital because of breathing difficulties and

required intensive care and ventilator support. Pulmonary hemorrhage

wasn't always apparent before breathing difficulties surfaced but

was detected once respiratory support began. Researchers suspected

that some element in the infants' home environments was responsible

for the symptoms because in several cases symptoms recurred when an

infant returned to his or her home.

In a case-control investigation begun in November 1994 by the

Centers for Disease Control and Prevention, researchers found

evidence that the geographically clustered infants' homes were

contaminated with S. chartarum, a mold not commonly found in home

environments. S. chartarum is known to produce several

trichothecenes, specifically satratoxins and roridin, as well as

phenylspirodrimanes, cyclosporin, and a newly discovered class of

compounds, the stachybocins. Researchers hypothesized that the

mycotoxins, in combination with other stressors in the infants'

environments such as tobacco smoke, caused the respiratory ailment

that claimed the lives of 12 of the 37 infants. " The primary problem

with indoor molds is that the health hazard is predominantly linked

to people who are atopic--that is, they tend to be allergic, " says

Dorr Dearborn, an associate professor of pediatrics and biochemistry

at the Case Western Reserve University School of Medicine in

Cleveland and one of the researchers associated with the ongoing S.

chartarum investigation. Says Dearborn, " What we're beginning to

realize more recently, which is not really well known in the medical

field, is that there are [indoor] molds--not just Stachybotrys, but

probably a larger list of them--that produce mycotoxins that can

have direct effects on health. This is still an area of both

speculation/conjecture and some knowledge, but it's an area of

active concern and with some research at least starting to be

generated. "

The precise mechanisms of the Stachybotrys mycotoxins are unknown,

explains Dearborn. The trichothecenes may be able to trigger or

aggravate an allergy problem directly, he says, but not through a

traditional immunoglobulin E (IgE) pathway. Typically, an allergic

response involves production of antibodies constructed from IgE

against the allergen and some form of inflammation (such as asthma).

Pestka's work to elucidate how deoxynivalenol induces nephropathy

has shown that, at low levels, the trichothecene induces

inflammatory mediators. This suggests a mechanism by which

mycotoxins, including those produced by Stachybotrys, may produce

airway disease or skin reactions without going through the typical

IgE mechanisms, says Dearborn.

Further information on Stachybotrys toxicity is gleaned from older

literature describing agricultural exposures to the mold in Eastern

Europe and northern Russia during the 1940s and 1950s. In this

literature, the effects include bleeding in the nose and throat

(although not the lungs), skin irritation, and altered white blood

cell counts. " The cellular mechanism of trichothecenes is well

established: they are potent protein synthesis inhibitors, " says

Dearborn. Inhibition occurs via a single binding site on the

ribosome, the cellular location of protein construction. Depending

on the specific trichothecene, construction breaks down during its

initiation, elongation, or termination stages, he explains. However,

he continues, the details behind their effects on the immune system

remain unknown.

Dearborn and his associates are currently in the early stages of a

five-year grant from the NIEHS to develop an infant model for S.

chartarum exposure as an outgrowth of their investigations into the

Cleveland outbreak of idiopathic pulmonary hemorrhage. " In the

Cleveland situation we're dealing with an epidemiological

association--that is, we've found [the mold] in the houses of the

cases more than we found it in the control houses. The link is not

absolute at all; it's simply an epidemiological link, " says

Dearborn. One of the scientists' immediate challenges comes in the

form of identifying a biomarker for exposure, a difficult task given

how rapidly the suspected toxins are metabolized. Finding a

biomarker is difficult because suspected Stachybotrys toxins are

rapidly metabolized and most people do not form antibodies in

response to the mold. The researchers' early experiments to

duplicate the disorder in infant animals have been promising. " What

we have shown is that if the spores of Stachybotrys are instilled in

the tracheas of young rat pups, they will develop pulmonary

hemorrhage. Initial results suggest that we are on the proper route

to develop an infant model for the disorder, " says Dearborn.

Areas for Further Research

The diverse spectrum of mycotoxins produced by S. chartarum

illustrates one of the more vexing issues in mycotoxicology:

mycotoxins usually occur in mixtures. As a result, researchers

recognize that interactions are possible although they are difficult

to characterize. There is particular interest in exploring potential

synergies, or interactions in which exposure to more than one

mycotoxin results in a multiplication, rather than an addition, of

risks. " The kinds of experiments that are necessary to elucidate the

nature of synergy are complicated, " says J. , a

professor of chemistry at Carleton University in Ottawa, Canada, who

recognized early on that the toxins produced by molds are typically

mixtures of toxins. Such experiments would require a lot of

resources that currently are not available, so investigating

potential additive effects or synergy between mycotoxins--or between

mycotoxins and other environmental factors--is, for the most part,

not a major research focus. Nevertheless, researchers have

commented, for example, on the potential interaction between

fumonisins and aflatoxin. For now, says , the most important

synergy that has been investigated with regard to mycotoxins is the

one that exists between aflatoxin and hepatitis B.

Another area of mycotoxin research that seems ripe for further

investigation concerns defining the subtler effects of individual

mycotoxins. In the area of veterinary toxicology, pinning down

information on such effects is an especially active area of

interest, according to Rottinghaus, a chemist in the

toxicology section of the University of Missouri Veterinary Medical

Diagnostic Laboratory in Columbia. " Those are what I call the gray

areas, " he says. " Everybody's done acute, subacute, and that type of

work, but it takes a lot more effort to get into these more subtle

changes. [With] a lot of these subtle things, you really wouldn't

have symptoms. It would be more of a performance- or immune-type

response effect that most people wouldn't see. " He offers an

example: " All of a sudden your animals might be sicker than they

normally are . . . or they [would be] off 5-10% in milk production,

or they don't gain [weight] quite the way they were supposed to. "

Rottinghaus points out that these symptoms can be attached to many

other factors, but it's hard to pin them to either mycotoxins or

alternative explanations.

Controlling Exposures and Mitigating Effects

The best means of preventing the health effects of mycotoxins is to

prevent exposure--a task more easily noted than achieved. In the

agricultural arena, postharvest control of storage fungi is handled

through proper drying and storing of grains. These measures are

accomplished with varying degrees of adequacy depending on the

available equipment, storage facilities, and other variables.

Success in preventing field contamination can be even more variable

owing to factors such as insect infestation, drought, or weather

events such as hail storms. Once crops are damaged, an opening

appears for fungal contamination. Whether or not fungi will exploit

that opening depends on other factors, including the prevailing

temperature, humidity, and water content. In some fields,

microclimates may exist so that one part of the field can be heavily

contaminated while neighboring sections are completely untouched.

Stachybotrys chartarum

Photo: Dorr Dearborn

Although use of antifungal agents and other chemicals is potentially

effective, researchers are investigating several strategies that

don't rely on chemical applications. " A number of the corn companies

are trying to develop hybrids that are resistant to the Fusarium

infection, " says Rottinghaus. According to Voss, researchers at the

USDA are exploring another avenue in investigations centering on

potential biological controls of fungal growth and toxin production

in corn plants. The idea behind their strategy is to use

nontoxigenic bacterial or fungal species as bioexclusion agents that

would outcompete fungi in the field and in storage. This technique

would only be used for corn destined for animal feed, and Voss

indicates that USDA researchers anticipate some commercial

applications of the technique within the next five years.

In some areas of the world, fungal control techniques are more

urgently needed. points out that in North America the

population experiences a relatively low risk from mycotoxins owing

to a diverse diet and the range of zones in which crops are

raised. " In North America, we produce large amounts of a crop, and

we only use a small percentage of it for human food. If we have a

bad year in Texas, it's unlikely it's [also] a bad year in Iowa. We

have the luxury, by and large, to pick and choose in terms of

excluding crops from our food system if it's necessary, " he

explains. For example, in 1996 wheat grown in Michigan, Maine, and

Ontario couldn't be used because it was heavily contaminated with

vomitoxin. However, because the commodity is also grown, albeit in

lesser quantities, in Alberta and the U.S. Pacific Northwest, buyers

could find supplies elsewhere. " In developing countries, that luxury

is not there, " says.

In recognition of this fact, researchers are attempting to find

other means of protecting populations from the health effects

associated with mycotoxin exposure. Much effort has been devoted to

applying such measures in AFB1-exposed populations. One technique

has been to promote vaccinations for hepatitis B in areas with high

AFB1 exposures. Another strategy explores the potential for altering

the metabolism of the toxic compounds. Recently, human trials were

conducted with oltipraz, a compound that interferes with the

mechanism of action of aflatoxin. The trials were published in the

February 1999 issue of the Journal of the National Cancer

Institute. " We'd like to believe from the data we have from the

oltipraz clinical trial that agents that can blunt the metabolism of

aflatoxin are certainly going to be important in terms of preventing

aflatoxin-mediated DNA damage, " says Groopman, who collaborated with

researchers in China on this study. " With the oltipraz intervention,

we found that we can modulate the metabolism of aflatoxins in people

and shunt the metabolism toward non-DNA-damaging species. If that

can be replicated by other dietary agents, that is probably going to

be a very important way of intervening in large populations, " he

concludes.

Efforts to control exposure to mycotoxins are certainly better today

than in ancient times, but they still are not perfect. For example,

the testing for mycotoxins such as aflatoxin only involves grains

that enter interstate commerce and thus doesn't protect people who

might consume highly contaminated locally grown crops. Other

populations may face greater risk simply because they consume higher-

than-average amounts of certain commodities or because they live or

work in poorly maintained buildings. Finally, research has primarily

emphasized dietary routes of exposure. Knowledge about the long-term

effects of other exposures is lacking. " The toxins that enter the

crops we use for food . . . [are] sort of a by-product of chemical

warfare that's going on at a microbiological level, " says .

There are lots of experiments to do, he muses, and not a lot of

resources to do them.

R. Barrett

---------------------------------------------------------------------

-----------

Last Updated: January 4, 2000