Information on 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 food-born 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:

• inhibition of protein synthesis, thought to be the most important effect (Ueno, 1983; Ueno et al., 1984; Tutelyan and Kravchenko, 1981)
• inhibition of DNA synthesis (Thompson and Wannemacher, 1984), which might contribute to their radiomimetic properties
• impairment of ribosome function (NAS, 1983; Coulombe, 1993; Tutelyan and Kravchenko, 1981)
• inhibition of mitochondrial protein synthesis (Pace et al., 1985)
• induction of reparable single strand breaks in DNA
• 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).

It has been shown, for example, that the effects of sustained low doses can accumulate to the clinical picture associated with alimentary toxic aleukia (Mayer 1953a, 1953 b; Lutsky et al., 1978). Or they can yield the more diffuse problems that Croft et al.(1986) and Jarvis (1985) reported: a case of ongoing illness for several years in a family of five, with recurring respiratory illness, flu syndromes, sore throats, diarrhea, cough, headaches, fatigue, and episodes of alopecia. One man had leg pains. Eventually, trichothecenes were identified in air ducts and ceiling material in the family's house. Material extracted from these areas was toxic to rats and mice. Croft cited other reports by Forgacs (1972) of toxin exposures producing similar symptoms with central nervous system andneuropsychiatric manifestations.[10]

Respiratory is high and comparable to parenteral injections. Oral and dermal lethal toxicities are lower but produce similar systemic effects (Creasia et al., Dose Effect).

Sources

Skin Exposures

5-50 ng in liquid Min. erythema dose (guinea pig, rat) Ueno/Wannemacher et al. (1983)

209 ng/cm2 in liquid Min. erythema dose (monkey) Wannemacher/Bunner et al. (1983)

1 µg/cm2 Irritation (guinea pig, rabbit) Fairhurst et al. (1987)

2 µg Vesication, skin injury Bunner (1983)

0.25 mg/kg Severe illness, diarrhea (monkey) Bunner et al. (1983)

1.5 mg/kg in DMSO LD50 (rat; mean time to death, 19 hr.) Wannemacher (1983)

4.2 mg/kg in methanol LD50 (guin pigs; time to death 190 hr.) Wannemacher et al. (1983)

Eye

1 µg Detectable corneal injury USAMRIID (1983)

2 µg Severe corneal injury, conjunctivitis Bunner (1983)

Respiratory

0.24 mg/kg (abs) Mouse LD50 Creasia et al. (1987)

0.05 mg/kg (abs) Rat LD50 Bunner et al. (1985)

0.6-2.0 mg/kg (abs) Guinea pig LD50 Wieser (1997, p. 661)

5,479 mg-min/m3 Guinea pig LD50 AD Little (1986)

200-1,800 mg-min/m3 Estimated LCT50 U.S. Army (1990)

Systemic Toxicity

500 µg/kg Estimated human LD50 U.S. Army (1990)

470 µg/kg intram. Rat LD50 Bunner et al. (1985)

1.17 mg/kg Rat LD50 Bunner et al. (1985)

650 µg/kgintram. Monkey LD20 Cosgriff et al. (1986)

790 µg/kg intraven. Monkey LD50 AD Little (1986)

850 µg/kgintram. Rat LD50 Chan and Gentry (1984)

111 mg/kgintram. Rabbit LD50 Chan and Gentry (1984)

Oral Toxicity

0.1-0.2 mg/kg Swine, emesis Busby and Wogan (79)

0.1-1.0 mg/kg Swine, diarrhea Ueno (1983a)

2.29 mg/kg Rat LD50 Bunner et al. (1985)

3.06 mg/kg Guinea pig LD50 AD Little (1986)

1.0 mg/kgb Male monkey LD100 Rukimi, Prasad, and Rao (1980)

1 5 mg/kg Estimated human LD50 U.S. Army (1990)

aNo primate data were located. bPer day for 15 days 1987; Bonomi et al., 1995; Wannemacher et al., 1993; Schiefer and Hancock, 1984).[11]

For less-than-lethal dosages and for all routes of administration, sequelae of leukopenia, thrombocytopenia, bleeding tendency, weakness, diarrhea, dyspnea, recurrent infections, vomiting, anorexia, and weight loss are expected. Other sequelae of acute exposures include prolonged rashes, joint pains and fatigue (Schultz, 1982), fever, chills, hypotension, confusion, somnolence, seizures, memory loss, hallucinations, and burning erythema (Belt et al., 1979; Murphy et al., 1978; Yap, et al., 1979; Diggs et al., 1978; Bukowski et al., 1982; Thigpen et al., 1981; Crossland and Townsend, 1984).

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).

Four stages were identified in the early Soviet Fusaria consumption incidents (a chronic oral exposure) (Mayer 1953a, 1953b):

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

Stage Four 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).

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

Among 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 among 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.