Aflatoxins

This family of related toxins is produced by the molds Aspergillus flavus and A. parasiticus, which commonly contaminate food grains before and after harvest. Their toxicity was recognized in the 1960s, and it was later appreciated that they are a significant health problem for domestic animals and humans. The toxins are stable and survive cooking. Attention has focused on chronic exposure and illness from oral intake, although there have also been acute effects (Steyn, 1995; Coulombe, 1993; Bonomi et al., 1995). This review concentrates on AFB1, the most toxic of the aflatoxin family, although the actual mixture Iraq weaponized is unknown. Aflatoxins show delayed acute toxicity (eight hours to several days) because most require metabolic activation (Daniels et al., 1990). However, most interest in aflatoxins arises from their carcinogenicity. They are implicated in the genesis of hepatocellular carcinoma, which is prevalent in tropical regions (Nigam et al., 1994, Groopman et al., 1996).

Aflatoxins do not appear to have attracted much of the military interest in toxins (SIPRI, 1973).[13] It was thus surprising when, after the War, Iraq informed the UN that it had produced aflatoxins and several trichothecene toxins (PAC, 1996a, 1996b; Zilinskas, 1997). Aflatoxins have, however, been mentioned as possibly enhancing the toxicity of trichothecene mycotoxins after the latter were recognized as military agents (U.S. Army, 1990; Schultz, 1982). Still, Iraq's placing aflatoxins in long-range missiles has surprised and puzzled analysts. Zilinskas (1997) offers three hypotheses:

a) The Iraqis discovered that aflatoxin possessed previously unknown properties useful in biological warfare.

b) The long-term potential for carcinogenesis was used to terrorize civilian populations.

c) Because aflatoxin is easy to produce, it was produced and deployed to meet toxin production quotas set by higher authorities.

This report examines the first and second hypotheses.

Although concerns about human exposure have focused on carcinogenic risks, acute aflatoxin toxicity has been recognized, primarily from oral exposure but also via the respiratory route (e.g., aflatoxin-contaminated grain dust) (Hendry and Cole, 1993; Zarba et al., 1992; Massey, 1996; Baxter et al., 1981, Autrup et al., 1993). Respiratory exposure would have been the most likely route in the Gulf. The available information indicates that the food and water supplies of U.S. forces in the Gulf theater were diverse but secure and did not present an opportunity for long-distance attack. Dermal exposure producing systemic toxicity from aflatoxins has not been described. Animal studies suggest that respiratory exposure is more toxic than oral exposure (Northup et al., 1995).

There is uncertainty about human clinical manifestations of low-dose respiratory exposure to aflatoxin, although both acute and chronic illnesses are expected. The symptom onset is delayed, and there is evidence of cumulative effects.

Although many humans are exposed to aflatoxin in food, gastrointestinal symptoms predominate; neuropathy, rashes, memory problems, and joint pain are not commonly reported. Aflatoxins do impair resistance to infection experimentally (Jakab et al., 1994; Raisuddin et al., 1993), although determining their role in increasing human infectious diseases has been difficult (Allen et al., 1992; Denning et al., 1995).

Weaponization

Information about military deployment of aflatoxin is apparently confined to the Iraqi experience, although the sources for this report were limited to the unclassified literature. Zilinskas (1997) was a member of the UNSCOM team that had access to Iraq and analyzed that country's biological warfare program, including toxin activity. It is evident that much of what the Iraqis chose to discuss could not be verified independently (Zilinskas, 1997).

Iraq began evaluating biological weapons in the late 1970s but began an earnest program in 1985. By April of 1991, Iraqi scientists had investigated the biological potential of five bacterial strains, one fungal strain, five viruses, and four toxins, while also developing two harmless bacteria for testing purposes. Major centers of development were Muthanna State Establishment (also the center of chemical weapon development) and Salman Pak, which became the biological warfare center, with production occurring at the Al Hakam Single Cell Protein plant. Virus research was conducted at an animal disease research station at Al Manal (Zilinskas, 1997).

Substantial efforts went toward weaponizing aflatoxin, botulinum, ricin, and perhaps trichothecenes. Generally, the Iraqis manufactured crude solutions of toxins. Iraq developed a method of producing aflatoxin using cultured rice as a growth medium. UNSCOM was told that some 2,200 liters of aflatoxin were produced at Salman Pak. Some toxin was stored after weapons were filled.

The weapons filled with biologicals (at Al Muthanna) included 250- and 400-pound bombs (60 to 85 liters of toxin solution) (Zilinskas, 1997). An unknown number of 122 mm rockets were filled with aflatoxin as were some ten Scud warheads (Zilinskas, 1997). The UN inspectors were told that tests were made using toxin-filled and stimulant-filled 122-mm rockets, but as far as they knew such weapons were not deployed. Iraqi munitions used a simple burster charge to open the walls and disseminate the agent. The Iraqis also possessed several hundred Italian-made pesticide dispensers suitable for biological dissemination by aircraft or land vehicle. A MiG aircraft was modified for unmanned operation and fitted with a 2,200-liter tank to disseminate chemicals or toxins. It must be understood that UNSCOM has not independently verified most information Iraq provided pertaining to toxins.

The amount of toxin needed to produce severe illness or death (2 to 4 mg/kg) via oral routes is greater than for many military toxins. This level of toxicity places it in the second order of toxicity classification on a six-category scale, in which 1 indicates "extremely toxic" (LD502 1 mg/kg), 2 indicates "highly toxic" (1-50 mg/kg), and 6 indicates "harmless" (Proctor and Hughes, 1978). Such chemicals as lewisite, DFP, and the organophosphate pesticides parathion and isosustox fall in this category (SIPRI, 1973). The uncertain late cancer effects provide an implausible military motivation for use.

Although Zilinskas (1997) was not certain about what military effects would result from use of aflatoxins, they are capable of producing death, seizures, respiratory injury, nausea, vomiting, and liver failure, which would be militarily significant (Chao et al., 1991; Northup et al., 1995; Jakab et al., 1994; Bourgeois, 1971a, 1971b). Inhaled aflatoxins in microgram amounts are highly immunosuppressive (Jakab et al., 1994) (milligrams would be needed for humans), but this effect would not provide the predictable effects weapon developers favor (use in conjunction with an infectious agent might be an exception).

In sum, the Iraqis were well informed about the effects of biological weapons and admitted conducting extensive field trials. A variety of fairly unsophisticated delivery means existed, as well as toxin stocks not placed in weapons. No information is available about bombing results on known biological warfare facilities. There is no indication that Iraq employed biologicals during the war, and there is no information about forward deployment of toxins in the theater. However, no detection system was deployed; no such system is available for aflatoxin, although there are mechanisms for detecting aflatoxin in the laboratory (Autrup et al., 1993; Harrison and Garner, 1991; Wang, 1996; Groopman et al., 1996; Ross et al., 1992). GAO (1997) suggested that aflatoxin exposure arising from U.S. attacks on chemical storage sites might have contributed to illnesses in Gulf War veterans but did not provide evidence to support the hypotheses.

The toxin is stable in the environment, is resistant to heat, and would be active after atmospheric transport from an attacked Iraqi depot. However, it is questionable how much significant toxicity would result after atmospheric dilution.

Mechanisms of Action

The concern about aflatoxin producing cancer in humans and animals has produced an extensive literature on the metabolism of aflatoxin and the biochemical reactions of the metabolites (Steyn, 1995; Coulombe et al., 1991, McLean and Dutton, 1995; Tutelyan and Kravchenko, 1981). Active metabolites act on several cell structures (e.g., mitochondria, lysosomes, endoplasmic reticulum), but the cancer concern has focused attention on the effects on the nucleus and DNA.

Activated AFB1 attacks nucleic acids with the formation of adducts that can act like point mutations, damaging DNA and impairing RNA and protein synthesis (McLean and Dutton, 1995). Proteins, including receptors and those with important intracellular functions, may also be nonspecifically but irreversibly bound by toxins, producing diverse loss of function (e.g., enzyme inactivation) (Tutelyan and Kravchenko, 1981).

Acute mycotoxin injury inhibits cellular energy production. The aflatoxins act on the electron transport system, interfering with the cytochrome system (Tutelyan and Kravchenko, 1981), depleting ATP, inhibiting ATPase, and causing mitochondrial swelling (Sajan et al., 1995). The effect of aflatoxin on the electron transport system may not require activation of the toxin. Recent studies draw attention to mitochondrial disease and injury, producing liver failure and associated disorders (e.g., Reyes syndrome), with brain and liver injury (Schafer and Sorrel, 1997). Carbohydrate and lipid metabolism are impaired, and hepatic glycogen stores are depleted, with a secondary rise in blood sugar. Lipids accumulate in the liver and fatty oxidation decreases, perhaps secondary to mitochondrial injury. These effects occur at levels lower than those producing RNA and growth effects (McLean and Dutton, 1995; Tutelyan and Kravchenko, 1981; Verma and Choudhary, 1995).

Activation.

Metabolic activation is required to produce toxicity from AFB1. After crossing the cell membrane, the molecule is activated by microsomal mixed-function oxidases involving the cytochrome P450 enzymes and nicotinamide adenine dinucleotide phosphate reductase (NADPH) and oxygen. The active and toxic AFB1 8,9-epoxide (Figure 4.3) is the result. This active molecule has a short half-life and binds to DNA and other structures in the endoplasmic reticulum. Other NADPH reactions can reversibly produce aflatoxicol, which can serve as a sink or source of AFB1 in the cell. The microsomal monoxygenase system may transform AFB1 into more polar molecules, such as AFM1, Q1, or P1, which can be eliminated by liver cells (McLean and Dutton, 1995). The situation is complex, and there are other concepts of toxicity involving more indirect mechanisms associated with membrane actions involving lipid peroxidases and aldehydes (Shen et al., 1994; Tutelyan and Kravchenko, 1981);

Detoxification.

Aflatoxins are also detoxified by mechanisms that deal with xenobiotics--leading to conjugation with glucuronic acid, sulfates, or glutathione. The major route for AFB1 detoxification is conjugation of the epoxide with glutathione (through glutathione S transferase) and subsequent excretion in bile (McLean and Dutton, 1995). This means that toxicity may vary depending on intracellular glutathione stores in various tissues, which can vary considerably with circadian effects or depletion by other factors--diet, smoking, alcohol, and medications (Tsutsumi and Miyazaki, 1994). Other aflatoxins appear to be primarily eliminated via glucuronide or sulfate conjugation. Various species differences in sensitivity to aflatoxins may reflect differences in detoxification mechanisms (McLean and Dutton, 1995).

Exposure-Effect Relationships and Clinical Manifestations

Gastrointestinal.

In Southeast Asia, intoxication occurs from ingestion of food, chiefly rice and noodles, that has been contaminated by the fungus. Outbreaks of illness attributed to oral intoxication by aflatoxin (Bourgeois et al., 1971b) have been reported in humans. Some 40 cases in Thai children were characterized by abrupt onset of coma or convulsions, fever, respiratory distress, vomiting, and death within 72 hours. Serum transaminases were elevated; prothrombin times were prolonged; and blood sugars were lowered. Pathology findings showed neuronolysis; cerebral edema; fatty infiltrations of liver, kidney, and heart; and lysis of lymphatic tissues (Chao et al., 1991; Chao, 1992).

In monkeys, a Reyes-like syndrome develops, with fatty degeneration of the liver (and encephalopathy) (Bourgeois et al., 1971a). Young monkeys were given 0, 1.5, 4.5, 13.5, or 40.5 mg/kg of AFB1 orally. Doses of 1.5 mg/kg were not fatal, and no unusual clinical signs were noted. Deaths began at 4.5 mg/kg, with others sick. All animals at higher levels died. Cough, vomiting, diarrhea, and coma were the key clinical findings. Laboratory findings were similar to those in the Thai children. Pathology was similar but also showed bile duct hyperplasia.

A well-studied outbreak occurred in Malaysia in 1988, with severe illness occurring in several towns that was eventually traced to noodles prepared from aflatoxin-contaminated grain. The noodles were also discovered to be contaminated with boric acid used as bleach. It took an average of eight hours (a range of 3 to 16 hours) from eating the noodles to the onset of symptoms. The illness began with vomiting (in 100 percent of cases), followed by seizures (82.4 percent), hematemesis (82.4 percent), fever (17 percent), diarrhea (23 percent), and abdominal pain. Liver and renal failure ensued, as did coma and respiratory failure. The outbreak killed 13 children, and another 45 persons had milder symptoms. The mortality rate was high, despite supportive modern medical care (Chao et al., 1991; Lye et al., 1995; Chao, 1992; Harrison and Garner, 1991). The estimated lethal amount was 2 mg/kg (Harrison and Garner, 1991).[15]

High doses result in multiple injuries. Those the Malay children sustained included gastric erosions, although these may have been related to the boric acid simultaneously ingested (Chao et al., 1991). Chao et al. made some effort to distinguish between the effects of aflatoxin and those of boric acid. They noted the patients lacked the "boiled lobster" skin changes of heavy boric acid poisoning but believed that the diarrhea, renal problems, and metabolic acidosis probably reflected boric acid effects. Overall, the researchers considered the toxicity to be primarily an effect of the aflatoxin. The most common findings in human and animal poisoning is liver injury (Lye 1991; Fernandez, Ramos, et al., 1995; Fernandez, Verde, et al., 1995), including macrovesicular steatosis (also a finding in Reyes syndrome), bile duct metaplasia, and centrilobular coagulative necrosis (Chao et al., 1991).AFB1 metabolizing enzymes are present in intestinal epithelial cells, which can sustain mild injury from low-dose AFB1 ingestion, impairing nutritional intake in animals (Guengerich et al., 1996). No follow-up reports are available to reveal what sequelae, if any, occurred in the survivors of acute exposures. Northup et al. (1995) indicate that the oral LD50 for guinea pigs is 1 mg/kg for AFB1.

Respiratory.

No estimates for an acute human lethal or incapacitating respiratory dose are available. There are also no descriptions of acute human or other primate respiratory exposures. The human lung does possess the enzymes necessary to activate AFB1 (Massey, 1996). At high levels of exposure, enough aflatoxin might be absorbed to produce the serious systemic illness seen in Malay children, with seizures, vomiting, coma, hepatorenal failure, and death. At low levels of exposure, there might be few obvious acute effects or only mild respiratory symptoms. However, there is some indication in animal studies that acute respiratory effects are possible at doses much lower than those required for dangerous oral intoxication (Cresia et al., 1987; Bunner et al., 1985). It may be that the Iraqis discovered some effects of that kind, and that is why they weaponized the toxin.

There has been concern about an increased risk of lung and liver cancer among humans chronically exposed to low-level AFB1 in grain dust at mixed oral and respiratory intake levels of 0.04 to 2.5 µg per week (Massey, 1996; Autrup et al., 1993; Coulombe, 1993). Mycotoxins have been shown to have mitogenic effects at levels well below clinical toxicity (Griffiths, Rea, et al., 1996). Other than the cancer risks, however, respiratory disease or systemic illness from chronic respiratory exposure has not been found (Coulombe, 1993); Autrup et al., 1993).

There have been some significant studies on animals, although caution must be exercised in generalizing these findings to humans. Sensitivity to aflatoxins varies tenfold among species because of metabolic differences and the balance between activation and detoxification mechanisms (Coulombe et al., 1991), and species vary as to the sites where aflatoxin is activated.

Experimental animals have shown a range of aflatoxin responses from mild inflammation to more striking illness with tracheal epithelial damage, alveolar injury, and pulmonary hemorrhage (Coulombe et al., 1991; Coulombe, 1993; Jakab et al., 1994). Guinea pigs, after a large aerosol exposure, developed hemorrhage and exfoliation of epithelial cells at six hours (Northup et al., 1995). Of particular interest, since the doses are in a range of potential military interest because of high potency, is a report that guinea pigs exposed for four hours to nanogram amounts by aerosol produced hemorrhage and exfoliation of respiratory cells (Northup et al., 1995). Intratracheal aflatoxin in rats appears in the blood in 3 to 12 hours (Coulombe et al., 1991). Minor bronchial mucosal damage occurred in rats that were intratracheally exposed to 300 µg/kg microcrystalline AFB1, but bronchiolitis occurred with intratracheal dust aflatoxin delivery (Coulombe et al., 1991).

Northup and Kilburn (1978; as cited by Hendry and Cole, 1993) reported tracheal-bronchial cell destruction in hamsters and guinea pigs from acute inhalation of aflatoxin.

Aflatoxin in rats was also retained longer when adsorbed on dust particles than when delivered in its microcrystalline form (Coulombe et al., 1991). Aflatoxins are known for their impairment of resistance to infections, so secondary respiratory and other infections might be expected. For example, macrophage function was impaired for two weeks following exposure to AFB1 aerosol in rats (16 µg/kg) (Jakab et al., 1994). Pulmonary pathology also occurs experimentally with oral aflatoxicosis, and there was some bronchopneumonia in the Malay outbreak (Fernandez, Ramos, et al., 1995; Fernandez, Verde, et al., 1995; Chao et al., 1991).

Rats given intratracheal aflatoxin B and G for 30 weeks developed carcinomas of the liver, intestine, and kidney (as cited in Hendry and Cole, 1993: Northup and Kilburn, 1978).

Nervous and Musculoskeletal Systems.

Aflatoxin is rapidly distributed to gray matter (Larsson and Tjalve, 1996), although no histopathologic descriptions of changes arising from aflatoxin exposure were found in the literature, other than the previously noted cerebral edema and neuronolysis noted in Thai and Malay children and in monkey experiments (Chao et al., 1991; Bourgeois et al., 1971a, 1971b). Animals and humans with high exposures to aflatoxin have seizures. The Malay cases showed widespread edema, with petechial hemorrhages in the white matter; however, these patients were on respirators for prolonged periods, and the brains were necrotic with impression of hypoxic encephalopathy (Chao et al., 1991). The mechanism for neurotoxicity is not clear, although brain cells have high metabolic rates, so disturbances in mitochondria and energy metabolism would be significant. No data have emerged to suggest that peripheral neuropathy is a problem arising from aflatoxin exposure, and there are no reports of musculoskeletal problems.

Studies in mice show that "nontoxic" low-level exposure to AFB1 reduced brain levels of serotonin and catecholamines (Kimbrough, Llewellyn, and Weekley, 1992). Although the clinical significance of this observation is unknown, it is noteworthy that this type of exposure to AFB1 affects these important neurotransmitters.

Cardiovascular and Hematologic.

No reports of characteristic cardiovascular findings were found. Cardiac hemorrhages have been described in cattle acutely poisoned by aflatoxin (Rajendran et al., 1992). Fatty degeneration of heart muscle (especially atrial and conduction systems) was seen in Thai children efficiently poisoned by AFB (Bourgeois, Olson, et al., 1971).

Hematologic problems, although noted, do not seem prominent. In vitro studies have shown dose-related inhibition of myelopoiesis in several marrow culture models (Cukrova et al., 1991; Dugyala et al., 1994). Aflatoxins impair phagocytosis by alveolar macrophages (Richard and Thurston, 1975). The impaired production of prothrombin in the liver in serious intoxications may contribute to the observed bleeding in other tissues.

Other Sites and Systems.

Because aflatoxins are soluble in DMSO (McLean and Dutton, 1995), it might be possible to deliver the toxin dissolved in DMSO through the skin. There is no information about acute or chronic cutaneous effects or hazards arising from cutaneous exposure.

Although the conjunctiva binds toxins, there is no information about inflammation or other acute or chronic toxic effects on the eye (Larsson and Tjalve, 1996).

Renal pathology is seen in acute toxicity, but does not appear to be a feature in chronic exposures. Autopsy data show swollen pale cortices with congested medullary regions. Aflatoxins M1 and M2 were found more often than B1 in renal tissue (Chao et al., 1991). Bourgeois, Olson, et al. (1971) noted fatty degeneration of kidneys with proximale tubule damage.

At levels below clinical illness, aflatoxins are immunosuppressive and impair humoral and cell-mediated immunity (Griffiths et al., 1996; Raisuddin et al., 1993; Cysewski et al., 1978; Dimitri and Gabal, 1996). Although acute effects of immunosuppression in animals reversed after two weeks (Jakab et al., 1994), longer exposure has resulted in loss of suppression of toxoplasma cysts (Venturini et al., 1996). It would not be surprising to see reactivation of quiescent infections, such as herpes.

Cross-Systemic and Chronic Effects.

Because of human health concerns, efforts are made to keep aflatoxins at low levels in food and milk. Interestingly, mice fed low levels of AFB1 and AFG1, within human exposure limits, showed signs of liver and kidney cytotoxicity, although species differences may play a role in this observation (Ankrah et al., 1993). Studies addressing chronic human exposure in occupationally exposed workers to unmeasured AFB in food show increased rates of liver cancer, impaired child health and development, and increased infections from long-term exposure (Groopman et al., 1996). No reports of neuropathy, chronic brain syndromes, skin problems, or arthropathy from chronic oral intake were available. Animal studies have found weight loss, illness, and reproductive problems from respiratory intake (Coulombe, 1993; Aulerich et al., 1993; Bonomi et al., 1995). Teratogenic effects have been observed (Raisuddin et al., 1993).

Combined Interactions

As noted before, there has been discussion of enhanced toxicity from combined exposure to trichothecenes and aflatoxins (U.S. Army, 1990). No reports of combined respiratory exposures were found. Some animal-feeding studies (chickens) found synergism (Huff et al., 1988), and others did not (Harvey et al., 1995), although the latter study showed some synergism in weight loss of a liver enzyme. We have not found a study that looked at synergism in acute respiratory exposures, which would be helpful in understanding the significance of this possible synergism.

What to Look for in the Gulf Context

The intended use of Iraqi aflatoxin weapons is unclear (Zilinskas, 1997). The toxin is stable enough to survive transport through the atmosphere and to persist trapped in dust, creating a secondary inhalation hazard. It is not clear that such transport and contamination occurred, but at low levels it would have been difficult to recognize or detect. The carcinogenic effects of aflatoxins take many years, and the risk from an acute exposure via missile attack does not seem enough to make it a credible objective.

There is insufficient information about respiratory effects in primates. Northup's finding of respiratory injury in animals with nanogram amounts of aflatoxin aerosol raises the possibility that respiratory toxicity is much greater than the better known oral toxicity, which might reflect the intended Iraqi use. Because the entire cardiac output passes the lungs, it might be possible to produce seizures, coma, and liver failure via the respiratory route.

Dramatic illness would have been noticed in the Gulf War if exposure to aflatoxin took place. There are no documented clinical reports of acute symptomatic lower-level human exposures, but extrapolation from animal studies suggests that low doses might produce respiratory irritation, nausea, malaise, and anorexia--symptoms not specifically associated with toxins or other chemical agents, where eye or skin problems would typically be expected. At levels comparable to those grain workers are exposed to, there might be no symptoms, although tissue and immune effects may occur.

Compared to most of the agents under review, it is hard to describe a "typical" aflatoxin case. As a result, it would be difficult to tie individual Gulf War illness cases to aflatoxin poisoning, even if it had occurred.

It is unknown what symptoms a combined low-level trichothecene and aflatoxin exposure would display. Although U.S. Army (1990) mentions synergistic effects, there are some data that prove a synergistic effect (Huff et al., 1988) and some that suggest that such a synergy does not always occur (Harvey et al., 1995). Studies of combined respiratory exposures would be helpful.

Summary, Conclusions, and Recommendations

Why Iraq developed weapon systems for aflatoxin remains speculative; respiratory toxicity at low levels and immunosuppression at low doses may provide hints, although there is little information about primate respiratory toxicity. Oral exposure in Malaysia led to deaths; if exposure at similar levels could be achieved through inhalation, pulmonary and systemic effects would be delayed, and there would have been no detection means (and no effective therapy). The agent, if spread in the vicinity of U.S. troops, would be stable enough to provide recurrent exposures. Although the estimated lethal dose of 2 to 4 mg/kg is not as toxic as some substances, it would be possible to create an aerosol that would deliver the 140 mg of toxin sufficient to kill a 150-pound person (if the Malay experience with children can be generalized). Of course, a full-blown outbreak of this nature would have been noted during the Gulf War. Lower levels of exposure might resemble acute oral exposure in animals, with vomiting, malaise, and nonspecific signs and symptoms.

There is no clear clinical picture that would make recognition of low-level aflatoxin exposure easy. However, there is also no information that aflatoxin was present in the Gulf War, and no descriptions of Gulf War illnesses resemble what might be expected from aflatoxin exposure.

Several steps might still be taken to assess the possibility of aflatoxin exposure in the Gulf. These include following up on a report that aflatoxin antibodies could be detected in exposed persons by measuring antibody levels in Gulf personnel and controls (Autrup et al., 1993). The antibody level in the Danish controls was low compared to that in Kenyans with high dietary intake, so finding antibodies in Gulf war veterans would not be conclusive proof of exposure to military toxin, since food exposure alone could promote antibody formation. Blood and tissue samples proximate to the Gulf deployment would be most useful, but care in study design and use of controls would be necessary. The antibody measurements are not routine but have apparently had substantial use.

Harrison and Garner (1991) detected aflatoxin and adducts in formalin-fixed pathology specimens a long time after the event in Malaysia. AFIP could consider analyzing some of its preserved tissues from Gulf cases for aflatoxins and adducts. Also, an analysis could be made of whether material from other Gulf War veterans shows adducts from aflatoxin. Adducts in Gulf War tissue material higher than those in controls would not prove a particular source of aflatoxin exposure but, if found, would require more research on this toxin.

Aflatoxin is sufficiently stable that it might still be detectable in clothing, equipment, filters, and mask canisters from the Gulf War, if they can be located. It would be useful to ask Malaysian health officials about any long-term effects in the less severe cases from the 1988 outbreak. Such a follow-up period would be about two years longer than the current Gulf War period of observation.

The aflatoxins are potent and poorly understood. They do not seem a likely explanation for the pattern of illnesses in Gulf War veterans, but it does at least appear possible to detect exposure of U.S. personnel to low levels of aflatoxins. Aflatoxins are a poorly understood agent, so further research on their possible military threats should be considered.

The respiratory toxicity of aflatoxins in primates and other species should be evaluated seriously, with some selective evaluation of combined toxicities (e.g., with trichothecenes or infectious agents). A better understanding of the mechanisms of central nervous system toxicity and immune suppression would be helpful (e.g., do aflatoxins alter responses to leishmaniasis, malaria, sand fly fever?).

Aflatoxins are known carcinogens.

The induction of cancer has generally been seen in populations with sustained exposures to fairly high dietary levels of the toxin after many years (and in some situations in populations with a high prevalence of chronic hepatitis B infections). A short (few weeks), low-level exposure to aflatoxins should have little risk of increased cancer because the incremental additional amount compared to the background level in Western diets would be small. [1] As noted earlier, anthrax and botulinum toxin are covered in Hilborne and Golomb (2000).

[2] OSRD (1946) says that "Ricin was recognized as a prototype of toxic protein materials of bacterial origin which were known to have even greater toxicity but which were less conveniently prepared and handled." (The reviewer assumes that the microbial toxin was botulinum toxin.)

[3] This favorable weather may not have always occurred during the air and ground war period of the Gulf War.

[4] This raises the possibility of cumulative toxicity from sustained low-level exposures in immune-compromised humans. The U.S. personnel in the Gulf region are presumed to have been immunocompetent.

[5] The animals were exposed to 1.2 µm particles in an aerosol of 128-353 mg-min/m3 (10 min) with absorbed dose calculated from respiratory parameters and impinger measurements.

[6] The larger dose here is the minimum lethal dose, the lowest amount that killed rabbits in LD50 tests--48 hours of LD50 0.54 µm/kg.

[7] The professor also observed chemical casualties in Iran, and his treatment recommendations were a subject of controversy in 1997 and 1998 letters in Lancet.