Trichothecene mycotoxins, specificity

This same experimental approach can be used to determine the appHcabiUty of the aDAS—AP to a competitive assay for DAS. As shown in Eigure 6, increasing amounts of free DAS were used to define the 50% inhibition level (ID q) of DAS for binding of two aDAS—AP conjugates to immobilized DAS. This approach was also used to determine the sensitivity of an EIA, as well as the specificity of the assay, as shown in Table 2. Increasing amounts of trichothecene mycotoxins closely related to DAS were added to microtiter plate wells containing a constant amount of prereacted DAS—aDAS—AP. After 30 min, excess toxin and any free toxin—aDAS—AP were washed out, and substrate was added. Quantification of the color produced was directly related to the abihty of the added toxin to displace aDAS—AP from the immobilized DAS, which is an indication that the aDAS also has an avidity for that toxin. 

Both no vaccine and no specific therapy are currently available for protection against any of the trichothecene mycotoxins and for poisoning by them. 

SPECIFIC SIDE GROUPS OF THE MOST ABUNDANT TRICHOTHECENE MYCOTOXINS... 

The trichothecenes make up a family of closely related chemical compounds called sesquiterpenoids (Figure 34-1). The structures of close to 150 derivatives of the trichothecenes are described in the scientific literature.35,43 The specific side structures of the most abundant of the naturally occurring trichothecenes are shown in Table 34-1. Because of its availability and relatively high toxicity, T-2 toxin has been the most extensively studied trichothecene mycotoxin. 

Studies with radiolabeled trichothecene mycotoxins suggest that the toxin interaction with cells is best viewed as (1) a free, bidirectional movement of these low-molecular-weight chemicals across the plasma membrane and (2) specific, high-affinity binding to ribosomes.51 Thus, further evidence indicates that the primary toxic effects of the trichothecene mycotoxins is caused by their properties as potent inhibitors of protein synthesis. 

Screening tests for the trichothecene mycotoxins are generally simple and rapid but, with the exception of the immunochemical methods, are nonspecific. A number of bioassay systems have been used for the identification of trichothecene mycotoxins.73 Although most of these systems are very simple, they are not specific, their sensitivity is generally relatively low compared to other methods, and they require that the laboratory maintain vertebrates, invertebrates, plants, or cell cultures. Thin-layer chromatography (TLC) is one of the simplest and earliest analytical methods developed for myco-toxin analysis. Detection limits for trichothecene mycotoxins by TLC is 0.2 to 5 ppm (0.2 to 5 pg/ mL). Therefore, extracts from biomedical samples would have to be concentrated 10- to 1,000-fold to screen for trichothecene mycotoxins. 

To overcome the difficulties encountered with the bioassays and TLC methods, immunoassays using specific polyclonal and monoclonal antibodies have been developed for most of the major trichothecene mycotoxins and their metabolites.73 These antibodies have been used to produce simple, sensitive, and specific radioimmunoassays (RIAs) and enzyme-linked immunosorbent assays (ELISAs) for the mycotoxins. In the presence of the sample matrix, the lower detection limits for identification of trichothecene mycotoxins by RIA is about 2 to 5 ppb73 and by ELISA, 1 ppb.74 We conclude that immunoassays are useful tools for screening biomedical samples for evidence of a biological warfare attack with trichothecene mycotoxins. 

No specific therapy for trichothecene-induced mycotoxicosis is known or is presently under experimental evaluation. Several therapeutic approaches have been evaluated in animal models. It is perhaps significant, however, that although experimental procedures for treatment of systemic exposure have been successful in reducing mortality in animal models, they have not been tested in primates. Thus, these treatments are not available for field use for humans exposed to trichothecene mycotoxins. 

No specific therapy for trichothecene mycotoxin poisoning is currently available. Skin decontamination with soap and water or the hypochlorite- (M258A1) or resin-based (M291) military decontamination kits can effectively remove toxin up to six hours after exposure, although none of them neutralize the toxin. Treatment of respiratory, dermal, and GI effects currently must be symptom based and supportive in nature. Superactive activated charcoal, for example, a common treatment for many orally taken poisons, has been shown to bind 0.48 mg T-2/gm charcoal in mice and improve survival rates significantly. 

As made evident in Table 2, immunoassays are also in routine use for analysis of mycotoxins, a group including the most dangerous and analytically elusive food-related toxins. In spite of the small structural differences within specific groups of mycotoxins (aflatoxins, ochratoxins, trichothecenes), assay specificity toward the other mycotoxin groups is always total. Determination of AEBi, toxin T-2, and ochratoxin A in a single extract from barley grains has been reported. Mycotoxins may be determined in crude extracts of various foodstuffs at concentration over 20 ng per kg. This detection limit can... 

GC/FTIR data have also contributed to the structure elucidation of other compounds of biological origin, such as mycotoxins, which are formed by fungal activity in food products under specific environmental conditions of moisture, temperature, and host. Trichothecene mycotoxins, secondary fungal metabolites produced by species of mold, are a natural contaminant of feedstuffs and food. Because they can be toxic to humans and animals, their detection is important. Sehat et al., utilized GC/MI-FTIR and GC/MS to analyze grains for these contaminants. 

Ohta, M., Matsumoto, H., Ishii, K. and Ueno, Y. (1978). Metabolism of trichothecene mycotoxins. II. Substrate specificity of microsomal deacetylation of trichothecene. jJ. Biochem. 84, 697-706. 

Lethality due to ingestion of food contaminated by trichothecenes has been reported in horses (Rodricks and Eppley, 1974), cattle (Hsu et al, 1972), and humans (Joffe, 1974). General clinical signs include emesis, food refusal and weight loss, dermal effects, and immune suppression with secondary infection. Clinical signs are dependent on the specific trichothecene involved, the dose, species, route of exposure, as well as the nature of the exposure. Spontaneous and experimental exposures may give somewhat different results, as can exposure to field contaminated materials, when compared to purified toxin. In the case of field contamination or experimental use of crude extracts, multiple mycotoxins, both known and unknown, may be present at the same time. 

The specificity of monoclonal antibodies make it possible to develop an analytical method for a single mycotoxin, such as aflatoxin Bi in maize and groundnut meal, or aflatoxin Mj in milk and milk products. Even within chemically closely related structures such as the Fusarium trichothecenes there is very little cross-reactivity between a monoclonal raised to a single toxin such as T-2 toxin and other members of the family. Thus, a monoclonal raised against 3-acetyl-deoxynivalenol showed negligible cross-reactivity with deoxynivalenol, nivalenol, or T-2 toxin. 

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