Toxins

Mycotoxins

Endotoxins

Fusariotoxin

Introduction

Fungi are ubiquitous to the environment and primarily saprophytic, using nonliving organic material as a nutrient source for growth and reproduction. Many of these saprophytes can colonize organic water-damaged building materials. During the digestion process fungi secrete enzymes into the nutrient source to break down complex compounds into simpler compounds, which are taken up by the fungi and digested. The digested nutrients are classified into two categories, primary and secondary metabolites. The primary metabolites consist of cellulose and other compounds that are used for energy to grow and reproduce. The secondary metabolites, called mycotoxins, are produced to give fungi a competitive edge against other microorganisms, including other fungi. There are over 200 recognized mycotoxins, however, the study of mycotoxins and their health effects on humans is in its infancy and many more are waiting to be discovered. Many mycotoxins are harmful to humans and animals when inhaled, ingested or brought into contact with human skin. Mycotoxins can cause a variety of short term as well as long-term health effects, ranging from immediate toxic response to potential long-term carcinogenic and teratogenic effects. Symptoms due to exposure to mycotoxins include dermatitis, cold and flu symptoms, sore throat, headache, fatigue, diarrhea, and impaired or altered immune function, which may lead to opportunistic infection. Historically, mycotoxins have been a persistent problem to farmers and the animal husbandry industry in Eastern Europe and developing countries. Recently, however, research has implicated many toxin-producing fungi, such as Stachybotrys, Penicillium, Aspergillus and Fusarium species, to indoor air quality problems and building related illnesses. Inhalation of mycotoxin producing fungi in contaminated buildings is the most significant exposure, however, dermal contact from handling contaminated materials and the chance of ingesting toxin containing spores through eating, drinking and smoking is likely to increase exposure in a contaminated environment. Recent advances in technology have given laboratories the ability to test for specific mycotoxins without employing cost-prohibitive gas chromatography or high performance liquid chromatography techniques. Currently, surface, bulk, food and feeds, and air samples can be analyzed relatively inexpensively for the following mycotoxins:

Aflatoxin

Aflatoxin is one of the most potent carcinogens known to man and has been linked to a wide variety of human health problems. The FDA has established maximum allowable levels of total aflatoxin in food commodities at 20 parts per billion. The maximum level for milk products is even lower at 0.5 parts per billion. Primarily Aspergillus species fungi produce aflatoxin.

Ochratoxin

Ochratoxin is primarily produced by species of Penicillium and Aspergillus. Ochratoxin is damaging to the kidneys and liver and is also a suspected carcinogen. There is also evidence that it impairs the immune system.

T-2 Toxin

T-2 Toxin is a tricothecene produced by species of Fusarium and is one of the more deadly toxins. If ingested in sufficient quantity, T-2 toxin can severely damage the entire digestive tract and cause rapid death due to internal hemorrhage. T-2 has been implicated in the human diseases alimentary toxic aleukia and pulmonary hemosiderosis. Damage caused by T-2 toxin is often permanent.

Fumonisin

Fumonisin is a toxin associated with species of Fusarium. Fumonisin is commonly found in corn and corn-based products, with recent outbreaks of veterinary mycotoxicosis occurring in Arizona, Indiana, Kentucky, North Carolina, South Carolina, Texas and Virginia. The animals most affected were horses and swine, resulting in dozens of deaths. Fumonisin toxin causes "crazy horse disease", or leukoencephalomalcia, a liquefaction of the brain. Symptoms include blindness, head butting and pressing, constant circling and ataxia, followed by death. Chronic low-level exposure in humans has been linked to esophageal cancer. The American Association of Veterinary Laboratory Diagnosticians (AAVLD) advisory levels for fumonisin in horse feed is 5 PPM.

Vomitoxin or Deoxynivalenol (DON)

Vomitoxin, chemically known as Deoxynivalenol, a tricothecene mycotoxin, is produced by several species of Fusarium. Vomitoxin has been associated with outbreaks of acute gastrointestinal illness in humans. The FDA advisory level for vomitoxin for human consumption is 1ppm.

Zearalenone

Zearalenone is also a mycotoxin produced by Fusarium molds. Zearalenone toxin is similar in chemical structure to the female sex hormone estrogen and targets the reproductive organs.

Other mycotoxins of clinical significance are as follows:

Citrinin

Citrinin is a nephrotoxin produced by Penicillium and Aspergillus species. Renal damage, vasodilatation, and bronchial constriction are some of the health effects associated with this toxin.

Alternariol

Alternariol cytotoxic compound derived from Alternaria alternata

Satratoxin H

Satratoxin H is a macrocyclic tricothecene produced by Stachybotrys chartarum, Trichoderma viridi and other fungi. High doses or chronic low doses are lethal. This toxin is abortogenic in animals and is believed to alter immune system function and makes affected individuals more susceptible to opportunistic infection.

Gliotoxin

Gliotoxin is an immunosuppressive toxin produced by species of Alternaria, Penicillium and Aspergillus.

Patulin

Patulin is a mycotoxin produced by Penicillium, Aspergillus and a number of other genera of fungi. It is believed to cause hemorrhaging in the brain and lungs and is usually associated with apple and grape spoilage.

Sterigmatocystin

Sterigmatocystin is a nephrotoxin and a hepatotoxin produced by Aspergillus versicolor. This toxin is also considered to be carcinogenic. Other mycotoxins include - Penicillic acid, roquefortine, cyclopiazonic acid, verrucosidin, rubratoxins A and B, PR toxin, luteoskyrin, cychlochlorotine, rugulosin, erythroskyrine, secalonic acid D, viridicatumtoxin, kojic acid, xanthomegnin, viomellein, chaetoglobosin C, echinulin, flavoglaucin, versicolorin A, austamide, maltoyzine, aspergillic acid, paspaline, aflatrem, fumagillin nigragillin chlamydosporol, isotrichodermin and many more. As discussed there are many mycotoxins that can cause adverse health effects and even death in humans. The synergistic effect of exposure to multiple mycotoxins simultaneously is very poorly understood. Even more poorly understood are the by-products of mycotoxin degradation, particularly under the influence of strong oxidizing agents such as sodium hypochlorite and/or ozone, agents frequently used or misused by remediation personnel in the industry. More research is required in this field to better understand the relationship of fungal contamination, mycotoxin production on building substrates and building related disease.

Endotoxins

Endotoxin is the name given to a group of heat stabile lipopolysaccharide molecules present in the cell walls of gram-negative bacteria that have a certain characteristic toxic effect. The lipid portion of each molecule is responsible for its toxicity and can vary between bacterial species and even from cell to cell. When inhaled, endotoxin creates an inflammatory response in humans that may result in fever, malaise, alterations in white blood cell counts, headache, respiratory distress and even death. It is common to the environment due to the ubiquitous nature of Gram-negative bacteria. Exposure to elevated levels of endotoxin primarily occurs through exposure to aerosols from specific reservoirs such as cotton mills, wastewater treatment facilities, air washers, humidifiers and any other occupational settings where Gram-negative bacteria can flourish.

Mycotoxins

In addition to their roles as irritants and allergens, many fungi produce toxic chemical constituents (Kendrick, 1992; Miller, 1992; Wyllie and Morehouse, 1977).  Samson and co-workers (1996) defined mycotoxins as "fungal secondary metabolites that in small concentrations are toxic to vertebrates and other animals when introduced via anatural route".  These compounds are non-volatile and may be sequestered in spores and vegetative mycelium or secreted into the growth substrate. The mechanism of toxicity of many mycotoxins involves interference with various aspects of cell metabolism, producing neurotoxic, carcinogenic or teratogenic effects (Rylander, 1999).  Other toxic fungal metabolites such as the cyclosporins exert potent and specific toxicity on the cellular immune system (Hawksworth et al., 1995); however, most mycotoxins are known to possess immunosuppressant properties that vary according to the compound (Flannigan and Miller, 1994).  Indeed, the toxicity of certain fungal metabolites such as aflatoxin, ranks them among the most potently toxic, immunosuppressive and carcinogenic substances known (ibid.).  There is unambiguous evidence that ingestion exposure as well as exposures by the inhalation pathway have been correlated with outbreaks of human and animal mycotoxicoses (Abdel-Hafez and Shoreit, 1985; Burg et al., 1982; Croft et al., 1986; Hintikka, 1978; Jarvis, 1986; Norbäck et al., 1990; Sorenson et al., 1987; Schiefer, 1986).  Several common mycotoxigenic indoor fungi and their respective toxins are listed.

Volatile Fungal Metabolites

During exponential growth, many fungi release low molecular weight, volatile organic compounds (VOCs) as products of secondary metabolism.  These compounds comprise a great diversity of chemical structure, including ketones, aldehydes and alcohols as well as moderately to highly modified aromatics and aliphatics.  Cultural studies of some common household moulds suggests that the composition of VOCs remains qualitatively stable over a range of growth media and conditions (Sunesson et al., 1995).  Furthermore, the presence of certain marker compounds common to multiple species, such as 3-methylfuran, may be monitored as a proxy for the presence of a fungal amplifier (Sunesson et al., 1995).  This method has been suggested as a means of monitoring fungal contamination in grain storage facilities (Börjesson et al., 1989; 1990; 1992; 1993).  Limited evidence suggests that exposure to low concentrations of VOCs may induce respiratory irritation independent of exposure to allergenic particulate (Koren et al., 1992).  Volatile organic compounds may also arise through indirect metabolic effects.  A well-known example of this is the fungal degradation of urea formaldehyde foam insulation.  Fungal colonization of this material results in the cleavage of urea from the polymer, presumably to serve as a carbon or nitrogen source for primary metabolism.  During this process formaldehyde is evolved as a derivative, contributing to a decline in IAQ (Bissett, 1987). 

Objectives of the current study

The present study was conceived with two primary objectives.  First, this investigation shall characterize the fungal biodiversity of house dust.  This work shall investigate correlations between dustborne fungal species, and examine the ecological similar of positively associated taxa based on the hypothesis that positively associated dustborne fungi are likely to share habitat characteristics.  From this, a second hypothesis follows that mechanisms that permit the entry or concentration a given species will tend to facilitate the entry of other positively correlated taxa.  A second objective of this research if to assess the extent of genotypic variability in two dustborne Penicillia, P. brevicompactum and P. chrysogenum.  The goal of this work shall be to examine the extent of clonality within these two species, and to determine if the observed patterns of genotypic variation support the current species concepts.

Mycotoxins of significance produced by indoor fungi

Mycotoxin Primary health effect Fungal producers

Aflatoxins Carcinogens, hepatotoxins Aspergillus flavus, As. parasiticus

Citrinin Nephrotoxin Penicillium citrinum, Pe. verrucosum

Cyclosporin Immunosuppressant Tolypocladium inflatum

Fumonisins Carcinogens, neurotoxins Fusarium moniliforme,

F. proliferatum

Ochratoxin A Carcinogen As. Ochraceus, Pe. verrucosum

Patulin Protein synthesis inhibitor, As. Terreus

nephrotoxin Paecilomyces variotii

Pe. expansum

Pe. griseofulvum

Pe. roquefortii

Sterigmatocystin Carcinogen, hepatotoxin As. nidulans

As versicolor

Chaetomium spp.

Trichothecenes Macrocyclic

Satratoxins Protein synthesis inhibitors Stachybotrys chartarum

Myrothecium spp.

Trichothecenes, Non-Macrocyclic

Deoxynivalenol Emetic F. cerealis

(vomitoxin) F. culmorum

F. graminearum

T-2 toxin Hemorrhagic, emetic F. sporotrichioides

carcinogen

Verrucosidin Neurotoxin Pe. aurantiogriseum group

Xanthomegnin Hepatotoxin, nephrotoxin As. ochraceus

Pe. aurantiogriseum group

Zeralenone Estrogenic Fusarium spp.

SOURCES:  Burge and Ammann (1999); Rodricks et al. (1977); Samson et al. (1996)

Fusarium Mycotoxins

"The genus Fusarium contains important mycotoxin-producing species that have been implicated in human diseases, such as alimentary toxic aleukia, Urov or Kashin-Beck disease, Akakabi-byo or scabby grain intoxication, and esophageal cancer. Many of these mycotoxin-producing species have also been implicated in several animal diseases, including hemorrhagic, estrogenic, emetic, and feed refusal syndromes, fescue foot, degnala disease, moldy sweet potato toxicosis, bean hulls poisoning, and equine leukoencephalomalacia. The interest in toxigenic Fusarium species is increasing world-wide due to the discovery of a growing number of naturally occurring Fusarium mycotoxins that have practical importance as threats to human and animal health."  quoted from Toxigenic Fusarium Species by Marasas et alia, Penn State U, 1984. Chemical Names of Fusarium Mycotoxins from Marasas et al. and other sources (Toxigenic Fusarium Species by Marasas et alia, Penn State U, 1984). Some of the names are redundant, and some are the result of research in different countries where two or more names have been given to the same compound, a common phenomenon in science.

4-Acetoxyscirpenediol

monodeacetylanguidin.

3-Acetyldeoxynivalenol

8-Acetylneosolaniol

4- or 15-Acetylscirpentriol

Acetyl T-2 toxin

Anguidin.

Avenacein +1

Beauvericin +2

Butenolide

Calonectrin

15-Deacetylcalonectrin

Deoxynivalenol

Deoxynivalenol diacetate

Deoxynivalenol monoacetate

Diacetoxyscirpendiol

Diacetoxyscirpenol

Diacetoxyscerpentriol

Diacetyldeoxynivalenol

Diacetylnivalenol

Dihydroxydiacetoxyscirpenol

Enniatins+1

Fructigenin +1

Fusarenon.

Fusarenon-X

Fusaric acid

Fusarinic acid

F-2. See Zearalenone

HT-2 toxin.

7"-Hydroxydiacetoxyscirpenol

1,4-Ipomeadiol

Ipomeanine

1-Ipomeanol

4-lpomeanol

Lateritin +1

Lycomarasmin +1

Moniliformin

Monoacetoxyscirpenol

Monoacetylnivalenol X

Monodeacetylanguidin.

Neosolaniol

Neosolaniolacetate

Neosolaniol monoacetate.

Nivalenol

Nivalenol diacetate

Nivalenol monoacetate

NT-1 toxin

NT-2 toxin

Rd toxin

Sambucynin +1

Scirpentriol

Solaniol

T-1 toxin

T-2 toxin

Triacetoxyscirpendiol

Triacetoxyscirpenol

Vomitoxin

Yavanicin +1

Zearalenol

Zearalenone

Mycotoxins reported from Fusarium oxysporum:

Diacetoxyscirpenol

Diacetylnivalenol

Fumonisin B1

Fusarenon-X

Fusaric acid

Moniliformin

Neosolaniol

T-2 Toxin

Zearalenone

Additional Mycotoxins

Fusarium Mycotoxins

Fusarium oxyspurum mycotoxins

Chemistry & Toxicology of the Fusarium mycotoxins

Mycotoxins in general

Mycotoxins are the toxic chemicals produced by fungi for a variety of reasons. These include to attack or gain access to hosts by helping to dissolve cell membranes, or as protective measures against encroaching organisms. The production of mycotoxins within the fungus depends on food sources and the particular enzymes of the fungus and other environmental factors.  Mycotoxins are usually not found in spores, but are generally produced in the next stage, that of mycelium.  Many mycotoxins, such as Mycotoxin T2 (Fusariotoxin) or the Amanita-toxins can be lethal to animals.  Others, such as Psilocybin, are entheogenic, producing altered states of consciousness that are usually associated with shamanism/religion.  Others, such as the ergot derivatives are used for migraine and post-partum hemorrhage. Still others, such as penicillin, Fusaric acid, and Wortmannin have antibiotic effects, and Zearalenone with anabolic effects, but which may or may not be beneficial to the host organism depending on the mode of administration and dose.  By definition, only  mycotoxin-producing fungi can be used as mycoherbicides to attack, colonize and kill plants.

The most-studied mycotoxins in Fusarium are toxic to both plants and animals.  Some have antibiotic properties.  The mycotoxins of Pleospora have yet to be identified, but we know from reports in the lab where it is being researched that it has toxic effects on humans.  After over a decade of work on EN-4 (a "coca-killing" strain of Fusarium oxysporum forma specialis erythroxyli), the USDA has neglected to examine strain EN-4 mycotoxins.  And by ignoring this research, an ARS spokesperson was still able to repeat the written USDA "talking points" mantra which state that EN-4 does not produce or contain mycotoxins dangerous to animals or humans to various members of the press. This claim is disavowed by her superiors, such as Eric Rosenquist, who candidly offers that the work on the safety of EN-4 as a mycoherbicide, including tests on its mycotoxins--have yet to be done.

In the absence of hard data on mycotoxins present in the Fusarium oxysporum and Pleospora papaveraceae strains being considered for use as mycoherbicides, we can only speculate on what these strains may contain. We also must caution the reader that fungi can produce different toxins and varying amounts of toxins depending on which media they are growing on, humidity, temperature, and light, among other variables. Even the USDA has published on this phenomenon: "Cultures of F. proliferatum established from these samples produced fumonisins when cultured on rice. They also produced other toxins, including moniliformin and beauvericin, which were not found in naturally-infected field samples of rice. It is not known why moniliformin and beauvericin were not found in field samples. There may be mechanisms by which viable rice kernels suppress synthesis of moniliformin and beauvericin by F. proliferatum, that are not operative in autoclaved rice cultures. A better understanding of the mechanisms by which mycotoxin production is controlled in Fusarium sp. may lead to methods to control these compounds in food and feed.". USDA has yet to persue this research.

However, here, for comparison's sake and taking the aforementioned caveats about the variability of Fusaria into consideration, we may examine the series of mycotoxins that have been already isolated from Fusarium oxysporum and other Fusarium species.

Chemistry and toxicology of the Fusaria mycotoxins:

The mycotoxins produced by Fusarium species are structurally quite varied.  Often, there is a series of  closely related compounds which can be identified as a group, such as the Trichothecenes which lack nitrogen in their structure and Fumonisins and Lycomarasmins, which posses amine functions.  Rather than approach this field by chemical category or structure, we shall resort to an alphabetical listing of the compounds by their most-used common names, as registered in the Merck Index, Twelfth Edition, which we will quote extensively here.

Fusarium mycotoxins may leach into the soil, causing damage to plants and animals through leaching even after the fungus is no longer active.  Indeed, a very real risk may be extrapolated for humans, also.

Trichothecene Mycotoxins

Trichothecene mycotoxins are produced by fungi (e.g., Fusaria, Trichoderma, Myrothecium, Stachybotrys); 60 are known. These were originally isolated as possible antifungal microbials or as antiplant agents. Analysis of trichothecene (and aflatoxin) exposures is complicated by their natural occurrence: Their presence alone does not prove a biological attack.

Iraq has admitted to possessing trichothecene mycotoxins and testing them in animals and has been accused of using them against Iran (UNSCOM, 1991, 1992, 1995; Zilinskas, 1997; Heyndrickx, 1984). The report of Iraqi possession of trichothecenes followed a considerable period of interest, attention, and controversy about their use in Southeast Asia (between 1974 and 1981, against Lao and Khmer populations by communist forces) and in Afghanistan (by Soviet forces) (Crocker, 1984; Haig, 1982; Schultz, 1982; Seagrave, 1981). Wannemacher and Wiener (1997), concluded that the Soviets and their clients have used trichothecenes, and the authors present a detailed review of the history of the subject and associated controversy. There may have been shortcomings in the epidemiological approaches (Hu et al., 1989). There were also many difficulties and inconsistencies in agent sampling, transport, and analysis.

These toxins, until discovered in Southeast Asian attack environments, had not been on the usual lists of potential toxin weapons (SIPRI, 1973). Analysts recognized that the toxins could produce the injuries encountered (Watson, Mirocha, and Hayes, 1984). Subsequent research identified properties of military significance, e.g., skin injury from nanogram amounts; eye injuries from micrograms; and serious central nervous system, respiratory, gastrointestinal, and hematological toxicity via multiple routes of exposure (Watson, Mirocha, and Hayes, 1984; Bunner et al., 1985; and Wannemacher and Wiener, 1997).

History

These mycotoxins have been poisoning people and animals for a long time. They grow well at low temperatures and frequently contaminate grain and other foodstuffs. They have been implicated in foodborne illnesses on several continents (Ueno et al., 1984). A large disease outbreak in the Soviet Union during World War II, which involved thousands and had high mortality, was eventually traced to the consumption of grain contaminated by Fusaria molds, which had been left in the fields over the winter. The disease, alimentary toxic aleukia, resembled a severe radiation injury with nausea, vomiting, diarrhea, leukopenia, hemorrhagic diathesis, and sepsis.

These toxins are also hazardous via other routes. Domestic animals and farmers manifested skin and respiratory irritation and systemic malaise from exposure to contaminated dusts and hay. Human illnesses have arisen from trichothecene mycotoxin contamination of houses and ventilation systems, resulting in so-called "sick building" syndrome (Croft et al., 1986; Jarvis, 1985; Smoragiewicz et al., 1993). One family so exposed was affected with nonspecific symptoms whose cause was not identified for months (Myrothecium and Stachybotrys were identified). For a time, several trichothecene mycotoxins were tested as anticancer agents in clinical trials (Thigpen et al., 1981; Bukowski et al., 1982; Yap et al., 1979; Diggs et al., 1978; Murphy et al., 1978; Goodwin et al., 1981). Some laboratory accidents have added to experience with human exposure (Wannemacher and Wiener, 1997). In addition, there is considerable information on the effects of trichothecene mycotoxins on economically important animals (Ueno et al., 1984).

Reports of communist attacks on Lao tribal people, and later on the Khmer, began in 1974 with aircraft and helicopter delivery of colored smokes, dusts, and droplets. People near these attacks had signs and symptoms that did not resemble known chemical warfare agents. Later similar attacks were reported in Cambodia and Afghanistan. Symptoms included vomiting, dizziness, seizures, hematemesis, respiratory distress, hypotension, and blisters. Survivors were ill for a long time with rashes, joint pains, fatigue, and memory problems (Haig, 1982; Schultz, 1982; Crossland and Townsend, 1984).

Investigative teams in refugee camps were puzzled, identifying a toxic epidermolysis without other expected findings from known chemical agents (House, 1979), but intelligence analysts recognized the similarities to trichothecene intoxication. Later, clinical examinations, autopsies, laboratory tests, and tissue samples showed trichothecene mycotoxins (and a propylene-glycol carrier) together with tissue damage compatible with trichothecene effects (Crocker, 1984; Watson, Mirocha, and Hayes, 1984; Rosen and Rosen, 1982; Stahl et al., 1985).

Chinese analysts attributed a higher toxicity to trichothecene mycotoxins than to nerve agents. They alleged that, between 1975 and 1982, 6,000 Laotians; 1,000 Cambodians; and 3,000 Afghans had died from attacks with what came to be known as "yellow rain" (Fang, 1983).

During the Iran-Iraq War, especially in the fighting around Majoon Island, colored smokes and powders were used against Iranian forces, perhaps reflecting combinations of agents. Although controversial in the scientific community, Heyndrickx (1984) found trichothecene mycotoxins in Iranian casualties who appeared to have sustained mustard injuries. Although other laboratories did not confirm these findings from the same material, Professor Heyndrickx argued that biological tissues had degraded the toxin over time.[7]

It is not known if, during the Gulf War, any of the Iraqi chemical and biological facilities hit by Allied fire contained trichothecenes. Trichothecenes are very resistant to environmental degradation and resist heat below 500¡F; hence, the production of effects after long-distance transport following explosive release is possible but unlikely because the chemical would be very diffuse by that time (U.S. Army, 1990; Wannemacher and Wiener, 1997; Trusal, 1985). However, no events described during the war closely correspond to known acute effects of trichothecene syndromes. Lethal effects require substantial doses (milligrams), but eye and skin irritation can occur at much lower levels (U.S. Army, 1990; Wannemacher and Wiener, 1997; Coulombe, 1993), raising the remote possibility that low-level exposures might have been misinterpreted as being due to some other cause.

Weaponization

Production using contemporary fermentation methods similar to those of brewing and antibiotic production is easy and inexpensive, and conventional bioreactors can readily produce tons of these agents (Wannemacher and Wiener, 1997). AD Little (1986, Ch. 4) described the conditions defining production. The large-scale production of Fusaria and trichothecenes for civil purposes in the former Soviet Union indicates the ease of large-scale production for other purposes (Buck et al., 1983). Formulations of T-2--one of the most potent trichothecenes--might also include polyethylene glycol, sodium lauryl sulfate, or dimethylsulfoxide (DMSO). These materials facilitate dispersal and handling of the toxin, possibly enhancing toxicity. Trichothecenes do not degrade to nontoxicity when exposed in the natural environment (for weeks at least) and are stable when stored. They can be delivered by mortars, artillery, free rockets, aerial bombs, and surface or aerial sprayers (Wannemacher and Wiener, 1997). Iraq possessed all the systems previously used to deliver trichothecenes.

T-2 is a skin-damaging agent of great potency (Bunner et al., 1985)--several hundred times more potent than mustards or lewisite (Wannemacher and Wiener, 1997). It is able to injure the eye in microgram amounts, which again indicates that it is more potent than mustards.

Toxicity by inhalation is comparable to mustards. NAS (1983) estimated that LC50 exposures of aerosols of 1 mg/m3 or surface contamination or LD50 of 1 g/m2 could readily be attained.

Trichothecenes readily result in vomiting, rather promptly at low concentrations, which might compromise the ability of exposed troops to use protective respirators. Other symptoms, including mild incapacitation, follow. Operationally, the persistence of trichothecenes makes them a threat even to military forces with protective equipment; Soviet troops in Afghanistan avoided operating in areas where these toxins were used (Fang, 1983). There are some indications that trichothecenes may have been used in combination with other agents in Southeast Asia and Afghanistan (Fang, 1983; Schultz, 1982).

Chemical and Physical Properties

The trichothecenes are classed as sesquiterpenes (Ueno, 1983). The members of this family of toxins vary depending on their side groups and include T-2, HT-2, nivalenol, deoxynivalenol, anguidine, diacetyoxyscirpenol (DAS), and crotocin.[8] When the toxins are extracted from fungal cultures, a yellow greasy residue remains. Had the various reported Asian attacks involved a crude extract containing some of that residue, the result might have been the yellow rain reported. The toxins are stable in air and light for weeks and can withstand heat; a temperature of 500¡F is required to destroy T-2 (Trusal, 1985; Wannemacher and Wiener, 1997).

These toxins can be inactivated with 3- to 5-percent hypochlorite solutions (Wannemacher and Wiener, 1997). The toxins are relatively insoluble in water but are soluble in acetone, chloroform, DMSO, glycols, ethanol, and other organic solvents. They have a peppery odor and negligible vapor pressure.

Detection

No military field detection systems currently deployed can detect trichothecenes, although laboratory techniques (e.g., antibody-ELISA, gas chromatography or mass spectroscopy, and thin-layer chromatography coupled to fluorimetry) have been used. Biological detection systems using animals are neither specific nor easy (Fontelo et al., 1983; Mirocha et al., 1984; Thompson and Wannemacher, 1984; Rosen and Rosen, 1982; NAS, 1983). Wannemacher and Wiener (1997), reviewing confirmatory procedures, indicated that mass spectroscopy is the procedure of choice, requiring little specimen "cleanup" and enabling detection of one part per billion (ppb) of toxin. More-complex systems being evolved may detect 0.1 ppb.

Toxicology and Toxicokinetics

Mechanisms of Action. The many mechanisms by which trichothecenes produce toxicity are varied, and their relative importance in producing illness is not fully understood (Coulombe, 1993). They include the following:

a) inhibition of protein synthesis, thought to be the most important effect (Ueno, 1983; Ueno et al., 1984; Tutelyan and Kravchenko, 1981)

b) inhibition of DNA synthesis (Thompson and Wannemacher, 1984), which might contribute to their radiomimetic properties

c) impairment of ribosome function (NAS, 1983; Coulombe, 1993; Tutelyan and Kravchenko, 1981)

d) inhibition of mitochondrial protein synthesis (Pace et al., 1985)

e) induction of reparable single strand breaks in DNA

f) immunosuppression, allowing secondary and opportunistic bacterial infections and possibly delayed hypersensitivity (Ueno, 1983; Yarom et al., 1984; Jagadeesan et al., 1982).

Trichothecenes react readily with thiol groups and, at low concentrations, inhibit thiol enzymes (e.g., creatine kinase, lactate dehydrogenase) (Tutelyan and Kravchenko, 1981; Ueno et al., 1984). They can be incorporated into lipid or protein elements of cell membranes. Tissue culture studies show alteration of membrane function (Coulombe, 1993; Pfeifer and Irons, 1985). Sulfhydryl effects in cell membranes are important in cell-to-cell interactions in the immune system. T-2 toxin induces cell membrane injury with hemolysis, apparently via a free-radical mechanism (Segal et al., 1983; Coulombe, 1993).

Metabolism may be more important in detoxification than in producing toxicity.

Unlike the aflatoxins that require metabolic activation, the trichothecenes are directly toxic without activation, as their prompt effects on the gastrointestinal mucosa with epithelial cell necrosis suggest (Busby and Wogan, 1979).

T-2 and other trichothecene toxins are deacetylated in the liver.

Metabolites are also toxic but less so than T-2 (Ueno et al., 1984). Carboxyesterases (-SH serine esterases) in liver microsomes hydrolyze T-2 to the less potent HT-2. These enzymes may be clinically important. Inhibition of this enzyme by paraoxon (an organophosphate pesticide) in subclinical doses increases the toxicity of T-2 in mice (Johnsen et al., 1986). Other potent inhibitors of this enzyme are tri-o-cresyl phosphate (TOCP, an organophosphate), eserine (a carbamate), and diisopropyl fluorophosphate (DFP, a weak organophosphate nerve agent) (OSRD, 1946). These all inhibit hydrolysis of T-2 (Johnsen et al., 1986). This raises the strong possibility that similar compounds, such as PB; low levels of nerve agent; or other carbamate or organophosphate insecticides might enhance the toxicity of T-2 or other trichothecenes at low levels.

Exposure-Effect Relationships

T-2 toxin and other trichothecenes are absorbed slowly (12 to 24 hours) via the intact skin but rapidly through abraded skin. DMSO or similar penetrants can increase the rate of absorption, but even then the systemic toxicity appears slowly (Bunner et al., 1985; Schiefer, 1984; Kemppainen et al., 1986a, 1986b; Solberg et al., 1990).

The rapid appearance of symptoms after respiratory exposure in humans, along with the results of animal inhalation studies, indicates rapid absorption and high retention of aerosolized T-2 toxin, with the respiratory tree retaining small amounts (Creasia et al., 1987).[9] Tritium-labeled agent and immunoperoxidase studies have also been used to follow the distribution and disposition of T-2 toxin (Pace et al., 1985, Lee et al., 1984). Intramuscularly injected agent is distributed to liver, kidney, lung, and other tissues within 30 minutes. The plasma concentration has a biphasic course, with half-lives of 1.8 and 50 hours for the two phases. T-2 toxin and metabolites concentrate in bile with evidence of enterohepatic circulation. The liver and kidney are the main organs for detoxification. Oral intoxication showed T-2 toxin in the gastrointestinal tract and kidneys, but not in the liver, reflecting rapid hepatic metabolism. The brain showed a rapid uptake to levels higher than plasma but below many other tissues, with a rapid fall to levels similar to plasma in six hours. One would expect trichothecenes to enter the brain readily, since they are lipophilic (Wang, Wilson, and Fitzpatrick, 1992).

Pathology and Pathophysiology

The clinical manifestations of trichothecene intoxication are derived from several sources. They are summarized here prior to more detailed treatment of individual organ systems.

Known effects from evidence other than the yellow rain attacks are nausea, vomiting, seizures, central nervous system dysfunction, chills, fever, hypothermia, hypotension, epithelial necrosis, myelosuppression, and gastroenteritis with hematemesis and melena (bloody vomiting and stools). In the yellow rain attacks, the Hmong victims were probably exposed by several routes, including dermal, respiratory, and oral (the last from swallowing larger particles trapped in upper airways and returned to the oropharynx by ciliary action (Wannemacher and Wiener, 1997). Vomiting was induced and lasted several days. There was a feeling of intense heat, itching and burning of the skin, dizziness, tachycardia, chest pain, headache, and decreased vision. Within hours, victims reported intense eye pain, red eyes, bleeding gums, and hematemesis. Trembling was common, and some patients had seizures. Severe itching ensued with the formation of small hard blisters, some of which were hemorrhagic, occasionally progressing to large bullae. Abdominal pain and bloody diarrhea continued (Watson, Mirocha, and Hayes, 1984).

Khmer yellow rain casualties had similar acute symptoms (Crossland and Townsend, 1984) with the following longer-term effects: intermittent weakness, anorexia, reduced memory and ability to concentrate, intermittent diarrhea, impotence, increased fatigue, cough and dyspnea, increased susceptibility to infection, and suspected increases in fetal abnormalities and spontaneous abortions (Haig, 1982; Schultz, 1982; Watson, Mirocha, and Hayes, 1984; Stahl et al., 1985; Crossland and Townsend, 1984). It must be noted that the Hmong cases with memory loss that Crossland described were not evaluated for the presence of toxins. These persons had undergone a harrowing experience, having been attacked, seen kinfolk die, fled, and become refugees. Severe apathy, confusion, and depression are common in survivors of natural or man-made disasters.

A limited autopsy was performed on a Kampuchean man injured in a toxic attack in February 1982, who died a month later having initially showed signs of recovery, then developing fever, jaundice, heoptysis, and anariax coma. Malaria was ruled out. The heart tissues showed interstitial myocardial hemorrhage and acute myocarditis, while the lungs showed only pulmonary edema. There was diffuse hepatitis with micronodular cirrhosis, as well as acute renal tubular necrosis. Tissues showed T-2 toxin in amounts ranging from 6.8 to 80 ppb, but there is little information with which to interpret the findings. The pathologist considered them to be compatible with mycotoxin poisoning (Stahl, Green, and Farnum, 1985).

4 stages identified in the early Soviet Fusaria consumption incidents

(a chronic oral exposure) (Mayer 1953a, 1953b):

Stage 1 begins within a few hours and lasts three to nine days. It consists of mild inflammation of the mouth and gastrointestinal tract, gastroenteritis, nausea, vomiting and diarrhea.

Stage 2

is a quiet period of two weeks or more with few symptoms even while contaminated grain was still being ingested. There were laboratory abnormalities in some patients, but most appeared well.

Stage 3

reveals the results of bone marrow aplasia with hemorrhagic diathesis, oral mucosal necrosis, and multiple infections.

Stage 4

is a period of convalescence requiring several months after ingestion stopped.

Grain elevator workers are in a complex environment with dusts, plant products, and trichothecenes. They frequently experience coughing, breathlessness, wheezing, fever, and dermatitis (Kemppainen et al., 1986b); similar problems occur in "sick" buildings (Hendy and Cole, 1993; Jarvis, 1985).

Respiratory

Rats, mice, and guinea pigs die rapidly from large respiratory exposures (1 to 12 hours) but show little sign of pulmonary injury, unlike direct effects on gastrointestinal mucosa (Creasia et al., 1987; Wannemacher and Wiener, 1997; Bunner et al., 1985). At low levels of respiratory exposure, coughing and upper respiratory irritation occur. Higher exposures produce pulmonary edema, collapse, hypoxia, and death within a few hours, or more indolent symptoms with later pulmonary hemorrhage, hypotension and shock, edema, or infections (Rukmini, Prasad, and Rao, 1980; Lutsky et al., 1978; Bunner, 1983; Bunner et al. 1985). Fifty Hmong survivors reported the following: smell of gunpowder or pepper (14 percent), rhinorrhea (28 percent), nasal itching (14 percent), sore throat (40 percent), aphonia (26 percent), cough (60 percent), dyspnea (52 percent), severe chest pain (52 percent), and hemoptysis (18 percent). Systemic signs (vomiting, tachycardia, hypotension, etc.) follow. Oral or intravenous exposures result in pulmonary edema, hemorrhage, consolidation, and secondary pulmonary infection.

Toxicity by the respiratory route may be influenced by the material used to suspend the toxin (Creasia et al., 1987). Fibrinous exudate may be seen, and pulmonary fibrosis was a late complication in some of the trichothecene cancer trials (Goodwin et al., 1981). In contrast to inhaled ricin, where effects are confined to the lungs, respiratory exposure produces much less pulmonary change and pronounced systemic toxicity.

Eyes

Conjunctivitis begins several hours after exposure, although the mechanism of the immediate visual disturbances is unclear. Corneal changes begin at 12 hours, with the peak effect in 24 to 48 hours. Blurred vision continues, with recovery from mild injuries in three to seven days. Hmong yellow rain victims reported eye pain and burning (68 percent), blurred vision (58 percent), and tearing (47 percent). Eyelid edema and scleral inflammation are associated with more-intense exposures. Corneal thinning can follow toxin exposure, with irregularities lasting up to six months (Bunner, 1983).

Skin

The skin responds to nanogram amounts of toxin with edema and inflammation. T-2 administered with DMSO to animals produced almost no local reaction (Bunner et al., 1985), but the systemic effects were substantial, although delayed, and cutaneous LD50s were elevated, compared to application without DMSO.

Dermal application can produce the same effects as oral administration: bone marrow, thymus, and lymphatic changes and gastrointestinal effects (Schiefer, 1984; Wannemacher et al., 1983).

In T-2 laboratory accidents, vesication has not been a problem

Despite decontamination, a burning sensation developed from 4 to 24 hours in the contact area, followed by numbness. In cancer trials, erythema, burning stomatitis, and alopecia were common (Schiefer and Hancock, 1984; Murphy et al., 1978; Bukowski et al., 1982; Diggs et al., 1978; Belt et al., 1979; Yap et al., 1979; Thigpen et al., 1981; Goodwin et al., 1981). Hmong survivors reported persistent burning sensations, with tingling, itching, and pain lasting several hours. Some numbness lasted two days to several months in some victims. Scattered erythema was noted after a few hours, but only 23 percent reported blisters. In some cases, large hemorrhagic bullae occurred, with underlying necrosis. Necrotic areas sloughed easily when corpses were moved (Wannemacher and Wiener, 1997). Sequelae include secondary infections, hyperpigmentation, and recurrent rashes.

Gastrointestinal

T-2 and other trichothecenes readily injure the rapidly dividing cells of the gastrointestinal tract. Tissue responses include edema, cytolysis, and sloughing, with loss of gastric epithelium and villus tips (Lee et al., 1984; Rukmini, Prasad, and Rao, 1980). The trichothecene DAS given intravenously showed marked gastrointestinal tract necrosis (Coppock et al., 1985) and pancreatic damage resulting in hyperglycemia. Some jaundice was seen in yellow rain victims. The liver is involved in detoxification, but liver failure is rare (Lutsky et al., 1978). Liver enzymes and amylase rise initially but return to normal in three to seven days (Bunner et al., 1985). As a later consequence, the bowel may become less resistant to bacterial penetration, which can increase susceptibility to infection (Lutsky et al., 1978).

Nervous System

The central nervous system effects are striking. Animals and humans exposed via the respiratory route show early central nervous system signs and symptoms. Symptoms reported from cutaneous exposures--burning pain followed by numbness--suggest that these toxins may directly affect the peripheral nerves.

The early and sustained vomiting suggests direct central nervous system effects involving chemotactic and vomiting centers. Hallucinations are a distinctive feature of trichothecene intoxications. Headaches, drowsiness, anxiety, confusion, and seizures occur, but their mechanisms have not been studied (Yap et al., 1979; Thigpen et al., 1981; Bukowski et al., 1982).

There are few autopsy reports. DAS-poisoned swine showed cerebral hemorrhages (Coppock et al., 1985), while other animal studies showed meningeal bleeding and scattered petechial hemorrhages (Ueno et al., 1984). Experimental studies show alterations in levels of hydroxyindoleacetic acid and seratonin in the brain, with regional norepinephrine increases. Trichothecenes make the blood-brain barrier permeable to mannitol, although not dextran (Wang, Wilson, and Fitzpatrick, 1992). Intracerebral administration of T-2 decreased learning in mice, and intraperitoneal administration disturbed both learning ability and memory (Umeuchi et al., 1996).

The descriptions of chemotherapy patients (Thigpen et al., 1981; Yap et al., 1979), home exposures (Croft et al., 1986), and yellow rain cases (Watson, Mirocha, and Hayes, 1984; Crossland and Townsend, 1984) convey a picture of neurotoxicity, with somnolence, confusion, tremors, depression, weakness, malaise, and memory problems (some of which resemble findings in some Gulf veterans). In the cases just cited, however, symptoms appeared promptly, and there were other conspicuous indications of exposure.

Cardiovascular, Lymphatic, Hematologic. Hmong yellow rain victims reported chest pain, sometimes crushing, along with weakness. Animals poisoned with T-2 develop tachycardia and later bradycardia. Hypotension occurs early and may persist for several days, sometimes proceeding to shock. Hypotension and orthostatic hypotension were common in chemotherapy patients (7 to 40 percent) (Yap et al., 1979; Thigpen et al., 1981; Murphy et al., 1978; Bukowski et al., 1982). Mucous membranes are bright red, reflecting vasodilation. Commonly, hemorrhagic foci are found throughout the myocardium (Ueno et al., 1984; Stahl et al., 1985), and the electrocardiogram may show a prolonged P-R interval and prolongation of the QRS and QT intervals, reflecting conduction system abnormalities and increased risk of arrhythmias.

Beginning with the alimentary toxic aleukia diagnoses, bone marrow and lymphatic system injury has been a consistent finding (Mayer 1953a, 1953b; Ueno et al., 1984). Cell culture studies show stem cells to be sensitive to T-2 toxin. Mycotoxins produce profound alterations in hemostasis, as noted in yellow-rain cases and documented by primate studies (Cosgriff et al., 1986). Prothrombin and activated partial thromboplastin times are increased early in intoxication from decreased coagulation factors. Lethal hemorrhage risk is greater because T-2 inhibits platelet aggregation (Yarom et al., 1984).

Other

There are clinical signs of muscle involvement. The Hmong complained of weakness, fatigability, tremors, and cramps. Animals show flaccid weakness after T-2 poisoning. The early elevation of serum creatine kinase could reflect muscle or cardiac injury, or both. Isoenzyme studies have not been reported (Bunner, 1983).

Impaired immunity and infection resistance is another effect of these toxins. The ability of leukocytes to kill bacteria is impaired (Yarom et al., 1984); immunoglobulin levels are depressed; and cell-mediated immunity is suppressed (Jagadeesan et al., 1982; Schiefer, 1984; Ueno et al., 1984).

Renal output decreases after T-2 intoxication, and the toxin is found in substantial amounts in the kidney. Observed tubular necrosis could be related to hypotension and liver disorders.

The endocrine effects of T-2 and other trichothecenes are not prominent. Adrenal cortical necrosis from T-2 exposure has been reported in rats (Thurman et al., 1986). Decreased spermatozoa production has been seen in several species.

Interactions

The literature on trichothecene interactions is limited. Combining aflatoxins and trichothecenes may increase toxicity (Schultz, 1982; U.S. Army, 1990). No reports emerged of studies examining combined inhalation exposures. There were indications of synergism in feeding studies of chickens (Huff et al., 1988). (However, a study of DAS and aflatoxin in lambs did not show any enhanced toxicity from combined oral exposures of these toxins (Harvey et al., 1995). There is a strong possibility that the severity of trichothecenes could be potentiated by exposure even to low levels of organophosphate pesticides, carbamate pesticides or pretreatments, or low levels of nerve agent, through inhibition of carboxyesterases involved in detoxification (Johnson and Read, 1987). Drugs inducing the increase of detoxifying enzymes, such as epoxide hydrolase or cytochrome P450, may favorably interact to decrease toxin severity. Such drugs as phenobarbital, metoclopramide, metochlopramide carbamazepine, metyrapone, and clofibrate have shown beneficial effects in animal models (Fricke, 1993; Wannemacher and Wiener, 1997).

What to Look for in the Gulf Context

Because of the high sensitivity of the skin and eyes to trichothecenes, injuries to these organs should be looked for in unit medical records. Conjunctivitis, erythema, burning skin, and blurred vision were followed by nausea, vomiting, and diarrhea might increase suspicion of trichothecene exposure.[12]

Summary and Recommendations

The trichothecenes are credible biological warfare toxins for some purposes. However, there is no proof or even a strong indication of their use against U.S. forces in the Gulf. With more concentrated exposure, hematological changes, seizures and other serious sequelae might have been expected.

Current information arises from clinically recognized exposures or laboratory research. Trichothecenes have multiple toxic effects with potential long-term consequences, such as central nervous system injury, immune suppression, and prolonged disability. The sequelae noted in the Hmong, e.g., long-term memory problems; the animal memory studies; and the story of the household exposure may resemble some features of the illnesses in Gulf War veterans, but the expected hematological alterations have not been reported among Gulf War patients. Furthermore, the Hmong effects resulted from substantial exposures with major short-term consequences. Little is known about the behavioral effects of sustained low-level exposures. The extreme sensitivity of the skin and eyes to T-2 and other trichothecenes makes it unlikely that delayed systemic illnesses in Gulf veterans represent a late effect of exposure to toxin "fallout." One would have expected an "epidemic" of painful dermatitis and conjunctivitis, as well as a number of other symptoms, which would have drawn attention to the exposure.

As in other cases, the AFIP should be consulted. The tests for trichothecenes are not routine, but have been used enough to be considered more than experimental. The AFIP might be consulted about the possibility of detecting trichothecene metabolites in tissue specimens obtained from the Gulf and immediately after. If used protective mask filters from the war period become available, it might be possible to analyze them for the presence of trichothecenes, which are very stable molecules. Had trichothecenes been used, it is possible that their toxicity might have been increased by interactions with nerve agents or PB, although this has not been studied explicitly.