Trichothecene Mycotoxins Chapter 34 TRICHOTHECENE MYCOTOXINS ROBERT W. WANNEMACHER, JR., PH.D.*; AND STANLEY L. WIENER, M.D.† INTRODUCTION HISTORY AND MILITARY SIGNIFICANCE Use in Biological Warfare The Yellow Rain Controversy Weaponization DESCRIPTION OF THE AGENT Occurrence in Nature Chemical and Physical Properties TOXICOLOGY AND TOXICOKINETICS Mechanism of Action Metabolism CLINICAL DISEASE Acute Effects Chronic Toxicity DIAGNOSIS Battlefield Diagnosis Confirmatory Procedures MEDICAL MANAGEMENT Individual and Unit Specific or Supportive Therapy Prophylaxis SUMMARY *Assistant Chief, Toxinology Division, U.S. Army Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick, Maryland 21702-5011 †Colonel, Medical Corps, U.S. Army Reserve; Professor of Medicine and Chief, Section of General Internal Medicine, Department of Medicine, University of Illinois College of Medicine, 840 Wood Street, Chicago, Illinois 60612 655 Medical Aspects of Chemical and Biological Warfare INTRODUCTION Mycotoxins, by-products of fungal metabolism, mental conditions. The distinguishing chemical have been implicated as causative agents of ad- feature of trichothecenes is the presence of a verse health effects in humans and animals that trichothecene ring, which contains an olefinic bond have consumed fungus-infected agricultural prod- at C-9, 10; and an epoxide group at C-12, 12.5 All ucts.1,2 Consequently, fungi that produce mycotox- trichothecenes are mycotoxins, but not all mycotox- ins, as well as the mycotoxins themselves, are po- ins are trichothecenes. This family of mycotoxins tential problems from both public health and causes multiorgan effects including emesis and di- economic perspectives. The fungi are a vast assem- arrhea, weight loss, nervous disorders, cardiovas- blage of living organisms, but mycotoxin pro- cular alterations, immunodepression, hemostatic duction is most commonly associated with the derangements, skin toxicity, decreased reproductive terrestrial filamentous fungi called the molds.3 capacity, and bone marrow damage.4,6 Various genera of toxigenic fungi are capable In this chapter, we will concentrate on T-2 myco- of producing such diverse mycotoxins as the afla- toxin, a highly toxic trichothecene that, together toxins, rubratoxins, ochratoxins, fumonisins, and with some closely related compounds, has been the trichothecenes.1,2 causative agent of a number of illnesses in humans The trichothecenes are a very large family of and domestic animals.1,2,4 During the 1970s and chemically related toxins produced by various spe- 1980s, the trichothecene mycotoxins gained some cies of Fusarium, Myrotecium, Trichoderma, Cepha- notoriety as putative biological warfare agents losporium, Verticimonosporium, and Stachybotrys.4 when they were implicated in “yellow rain” attacks They are markedly stable under different environ- in Southeast Asia.7–11 HISTORY AND MILITARY SIGNIFICANCE Fungi that produce trichothecenes are plant sprays delivered by T-28 aircraft (1979–1981) against pathogens and invade various agricultural products Khmer Rouge troops. The chemical munitions were and plants. Since Fusarium and other related fungi supplied by the Soviets and delivered by North infect important foodstuff, they have been associ- Vietnamese or Laotian pilots. In Afghanistan, the ated worldwide with intoxication of humans and chemical weapons were delivered by Soviet or Af- animals. Thus, these fungi have potential as bio- ghan pilots against Mujahidin guerrillas (1979– logical weapons. 1981). Lethality of the attacks is documented by a minimum of 6,310 deaths in Laos (from 226 attacks); Use in Biological Warfare 981 deaths in Kampuchea (from 124 attacks); and 3,042 deaths in Afghanistan (from 47 attacks).7 From 1974 to 1981, toxic agents were used by the Trichothecenes appear to have been used in some Soviet Union and its client states in such Cold War of these attacks. sites as Afghanistan, Laos, and Kampuchea (Cam- The air attacks in Laos have been described as bodia). Aerosol-and-droplet clouds were produced “yellow rain” and consisted of a shower of sticky, by delivery systems in the Soviet arsenal such as yellow liquid that sounded like rain as it fell from aircraft spray tanks, aircraft-launched rockets, the sky. Other accounts described a yellow cloud bombs (exploding cylinders), canisters, a Soviet of dust or powder, a mist, smoke, or insect spray– hand-held weapon (DH-10), and booby traps. Air- like material. Liquid agent rapidly dried to a craft used for delivery included L-19s, AN-2s, T-28s, powder. In Laos, 50% to 81%7 of attacks involved T-41s, MiG-21s (in Laos) and Soviet MI-24 helicop- material associated with a yellow pigment. Other ters (in Afghanistan and Laos). attacks were associated with red, green, white, or Attacks in Laos (1975–1981) were directed against brown smoke or vapor. More than 80%7 of attacks Hmong villagers and resistance forces who opposed were delivered by air-to-surface rockets; the remain- the Lao People’s Liberation Army and the North der, from aircraft spray tanks or bombs. Intelligence Vietnamese. In Kampuchea, North Vietnamese information and some of the victims’ descriptions troops used 60-mm mortar shells; 120-mm shells; of symptoms raised the possibility that chemical 107-mm rockets; M-79 grenade launchers contain- warfare agents such as phosgene, sarin, soman, ing chemicals; and chemical rockets, bombs, and mustards, CS, phosgene oxime, or BZ may also 656 Trichothecene Mycotoxins have been used. These agents may have been used Russians have not recently denied such use but have in mixtures or alone, and with or without the declined to discuss the subject. trichothecenes. In addition to the evidence stated above, elevated Unconfirmed reports have implicated the use levels and naturally rare mixtures of trichothe- of trichothecenes in the 1964 Egyptian (or Rus- cene toxins were recovered from the surfaces of sian) attacks on Yemeni Royalists in Yemen12 and in plants, fragments of plastic, and rocks in areas at- combination with mustards during chemical war- tacked9,11,15,16; and were detected in the blood of fare attacks in the Iran–Iraq War (1983–1984).13 Ac- attack survivors and the tissues of a dead casu- cording to European sources, Soviet–Cuban forces alty.10,15 Control samples that were taken (a) from in Cuba are said to have been equipped with myco- an environment that had not been attacked, and toxins, and a Cuban agent is said to have died of a during another season of the year,15 and (b) from hemorrhagic syndrome induced by a mycotoxin Hmong who had never been exposed to an attack agent.14 were consistently negative. The evidence that trichothecenes were used in The Yellow Rain Controversy Southeast Asia has been challenged: questions have been raised about the interview methodology used Actual biological warfare use of trichothecenes by U.S. Army physicians and U.S. State Department in Southeast Asia and Afghanistan is strongly sup- personnel in Hmong refugee camps in Thailand to ported by the epidemiological and intelligence as- obtain descriptions of the attacks. Some inconsis- sessments and trichothecene assays, although re- tencies of specific individuals’ stories were demon- ports in the open literature have discounted this strated, but the frequency of unreliable information contention. An article written by L. R. Ember,15 has not been reported and is unlikely to be large published in 1984 in Chemical Engineering News, is enough to discredit all witnesses.15 Symptom de- the most exhaustive and authoritative account of scriptions are generally consistent with known the controversy surrounding the use of tricho- trichothecene effects. thecene mycotoxins in Southeast Asia during the The paucity of positive evidence of the presence 1970s. of trichothecenes (5 positive environmental and 20 The United States government, its allies, and positive biomedical samples) has been used to chal- journalists exhaustively studied the possibility that lenge the belief that biological warfare attacks oc- yellow rain attacks had occurred, based on evi- curred, since only 10% of samples were positive. dence7,14,15 such as the following: However, 32% of samples from victims were posi- tive, a value too high for natural causes (eg, food • interviews of Hmong survivors of and eye- contamination) to be used as an explanation, since witnesses to lethal yellow rain attacks in 98% of controls in nonattack areas of Thailand were Laos, who provided consistent descriptions negative.17 The 2% of samples that were positive of the episodes; could represent either a nonspecific result or low- • interrogations of a defecting Laotian Air prevalence food contamination. The paucity and Force officer and North Vietnamese ground type of control samples have also been questioned. troops, who corroborated the descriptions Some experts18–21 have claimed that yellow rain of attacks and admitted using the chemi- was not a biological warfare attack at all, but that cals; the yellow residue was caused by showers and de- • interrogations of prisoners of war, who ad- posits of bee feces—the result of massive bee mitted being involved in attacks where un- swarming and cleansing–defecation flights over conventional weapons were used (ie, in some areas of Southeast Asia. The presence of pol- Afghanistan); len in bee feces and some samples has not only • laboratory confirmations of Soviet use of added confusion18 but is also the supporting evi- chemical agents, and dence used by the skeptics. It is important