Animal Assays

Vertebrates. Laboratory animals, especially mice and rats, have been successfully used for the detection and isolation of mycotoxins including aflatoxins, zearalenone, rubratoxins, and aflatrem (157). Advantages of using mice are that they are small, commercially available, and relatively inexpensive. Ducklings and chickens have been used successfully to detect ochratoxin A (172), a-cyclopiazonic acid (173), tremorgenic mycotoxins (174-176), monilifor-min (177), and some trichothecenes (178). One of the most sensitive bioassays for aflatoxin is the production of liver carcinoma in rainbow trout (179). However, the test is difficult to run and is impractical as a screening method because it takes more than four months to complete (157).

Several specific animal tests exist for the trichothecene mycotoxins. In the skin assay, a test solution is applied to the back skin of an experimental animal such as a rabbit, rat, or guinea pig, and the area is monitored for skin lesions. Sensitivities for this bioassay have been reported to be 0.1 jUg using T-2 toxin (180). Another trichothecene bioassay involves the rejection or acceptance of drinking water by mice (181). The test sample is dissolved in distilled water that is presented to mice. The volume of the contaminated water consumed by the animals is compared with the volume of control water consumed. A positive test occurs when the mice refuse to drink, indicating the presence of trichothecene.

A specific bioassay for zearalenone is based on the hy-perestrogenic effects of the mycotoxin (157). Swine are the most sensitive animals, although dairy cattle, lambs, chickens, turkeys, and laboratory animals (rats, mice, guinea pigs, and monkeys) are also affected. The rat uter-

otopic bioassay has been used as an assay for zearalenone if other analytical methods are unavailable (182).

The only vertebrate bioassay adopted by the AOAC is the chick embryo assay also known as the CHEST (chick embryo toxicity screening test) assay (157). The assay involves injecting test solutions into the air cell of fertile chicken eggs. Toxicity of the test solution is determined by comparing the mortality of the embryos injected with the test substance with that of the undosed controls. The CHEST assay has been used to determine toxic effects of individual mycotoxins (183,184) as well as combinations of several toxins (185). Overall, the method is simple, inexpensive, and sensitive, but it is nonspecific and prone to false positives (157). The reproducibility of the CHEST assay depends on the type and volume of carrier solvent, the site of injection, and the observation period (186). The sensitivity of the CHEST assay is in the 0.1- to 100-/;g range (158).

Factors that must be considered when using whole animal bioassays include the method of administration and the vehicle for administration (157). Oral routes of administration are the most valid since they simulate the processes that occur during ingestion, digestion, and absorption of the mycotoxins. Ideally, the test material is administered by feeding ad libitum in an inert carrier. Because of solubility problems, some toxins cannot be readily formulated in inert carriers. In addition, carriers can be toxic themselves or affect the absorption process (157).

Invertebrates. Several invertebrates (brine shrimp, protozoa, planaria, mollusks, and insects) have been screened for their ability to detect mycotoxins. The brine shrimp (Ar-temia salina) bioassay is one of the first and most widely used biological screening methods for mycotoxins and has been used to screen aflatoxin (187), trichothecenes (188), ochratoxin A (189), and fumonsin (184). The sensitivity range of the assay for 10 trichothecenes ranged from 0.04 to 0.4 jug/mL, with T-2 toxin being the most toxic (188). Attractive features of the assay include its sensitivity, simplicity, rapidity, and low cost. A disadvantage of the brine shrimp assay is that this organism is sensitive to many compounds present in normal foods and feeds, resulting in a high percentage of false-positive bioassay results (157).

Tissue Cultures and Cytotoxicity. Numerous vertebrate tissue and organ cultures have been used for bioassays for mycotoxins, including calf kidney cells; embryonic lung cells; rabbit reticulocytes; HeLa cells; human fibroblasts; and rat kidney, liver, and muscle cells (157). Cytotoxicity studies involve incubating the test substance with cell cultures for various periods of time. Cytological, morphological, and biochemical methods are used to evaluate toxicity (157). A mutagenicity/carcinogenicity assay using primary rat hepatocytes has been useful for determining if mycotoxins and other compounds induce chromosomal aberrations and micronuclei (190). Advantages of tissue culture and cytotoxicity tests are that they are sensitive, relatively inexpensive, and readily available. Several drawbacks include a lack of specificity and the fact that they do not take into account ingestion, digestion, and absorption of the toxin (157).

Plant Assays

Use of plants (whole, tissues) to detect mycotoxins has had limited application. However, there are several cases where plants have been excellent sensors for mycotoxins. Plant bioassays have an advantage over whole animal studies in that they are easier and less expensive to conduct. Plant bioassays have been used in the past to detect and isolate plant growth regulators. Since some mycotoxins also affect plant growth, whole plants and plant tissues are also used for mycotoxin screening. Malformin A, mon-iliformin, diacetoxyscirpenol, fusaric acid, and Alternaria alternata toxins are examples of mycotoxins that have been screened using bean, corn, rice, wheat, jimsonweed, and tobacco plants (191—193). The wheat coleoptile bioassay has been a useful tool for detecting chaetoglobosin K (194) and is especially sensitive to the 12,13-epoxytricho-thecenes (157).

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