MOM 102 5

Letters in Applied Microbiology 1990,10, 155-160

An appraisal of bioassay methods for the detection of mycotoxins-a review A . E . B U C K L E& M . F . S A N D E R SMicrobiology Department, ADAS Central Science Laboratory, London Road, Slough, Berks SL3 7 H J , U K Received 5 M a y 1989 and accepted 19 M a y 1989

B U C K L EA, . E . & S A N D E R SM, . F . 1990. An appraisal of bioassay methods for the review. Letters in Applied Microbiology 10, 155-160.

detection of mycotoxins-a

The literature on bioassay methods for mycotoxin detection has been reviewed. An outline of the range of bioassay methods is given and the role of cytotoxicity tests in particular has been emphasized.

The desirability of challenging a living system with any potentially toxic substance to assess its biological activity has led to the development of many different types of bioassay. The use of bioassays for detecting mycotoxins has been extensively reviewed by Watson & Lindsay (1982) who cite 78 papers covering 37 mycotoxins. Bioassays for mycotoxins have been developed using micro-organisms, plants, crustacea, fish, birds, mammals and organ and tissue cultures derived from animals. Many of these bioassays have been developed because toxicity tests on mammals are extremely expensive and require special laboratory facilities (Bul et a/. 1981). This has promoted the use of alternative bioassays permitting effects to be observed at the cellular or subcellular level (Engelbrecht & Purchase 1969; Kuroki et al. 1979). A mycotoxin bioassay may be used to elucidate the action of a mycotoxin at the biochemical level or to determine its effect on a whole animal. These two basic types of bioassay require different cell types. For example, Meneghini & Schumacher (1977) looked at the inhibition of DNA synthesis by aflatoxin B, in African green monkey cells. This was a relatively straightforward procedure, the end point, i.e. inhibition of DNA synthesis, was noted and further results indicated that aflatoxin B, blocked initiation of replication rather

than elongation. This type of bioassay may be used to gather information on biochemical processes within a cell. Bioassays are also used to demonstrate toxicity to a whole animal (Bodon & Zoldag 1974). The end point for this type of assay is difficult to extrapolate to the whole animal. The type of biological material used is also important, for example continuous cell lines (those having the apparent potential to be passaged indefinitely in oiuo) have lost some of their original biochemical functions found in the tissue from which they were derived. This type of cell would often be unsuitable for predicting some toxic effects in animals. Umeda (1971) showed that primary rat liver parenchymal cells were more susceptible in their reaction to hepatotoxic mycotoxins than other cells. These primary cells (derived directly from the particular organ or tissue of the host organism) retain most of their original functions and are often better able to demonstrate specific organ related effects which might be important to the whole animal. An excellent example of this was given by Scaife (1971) who exposed rat liver cells, slices or liver in uiuo, to aflatoxin B,. He found that DNA synthesis was rapidly inhibited in the liver cells while continuous cell lines were more slowly affected. He also stated that liver cells convert aflatoxin B, to a more potent cytotoxin which can then affect non-

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susceptible cells. He postulated that this may explain the susceptibility of liver to tumorogenesis by aflatoxin. This example shows the care which must be taken in the selection of bioassay systems for a meaningful extrapolation to be made to a whole animal. Meisner & Selanik (1979) also used tissue slices and demonstrated a reduction of glucogenesis from pyruvate in kidney cortex slices from rats fed ochratoxin A. Other examples of organ cultures are mentioned later. Cytotoxicity tests which are performed on organ and tissue cultures have many advantages over using animals. They are more convenient and are not controlled, unlike experiments on animals which require a Home Office licence. They can produce a result within 2 days and tend to be more sensitive to most mycotoxins than animals. Cell cultures can be kept viable under liquid nitrogen when not required or they can be maintained indefinitely by repeated subculture in the laboratory. Cytotoxicity tests generally possess a broad spectrum of sensitivity and are able to detect substances which are potentially injurious to animal cells. In their simplest form cytotoxicity tests are performed by administering a sterile chemical extract obtained from the material under test directly to a tissue culture. The presence of a toxic substance will impair or inhibit the biochemical activity, e.g. protein or DNA synthesis of the cell. This will be manifested by the cessation of cell growth or division and ultimately lead to cell death.

Cytotoxic Effects of Mycotoxins The cytotoxic effects of many mycotoxins have been investigated in a variety of cell cultures of animal and human origin. Some of these studies are summarized in Table I. Although most of the cell cultures tested were sensitive to many of the mycotoxins examined few of them have been applied to screening foodstuffs and fungal cultures for mycotoxins. One of earliest reported applications of a cytotoxicity test was based on the inhibitory effect that some mycotoxins have on protein synthesis by monitoring the uptake of ['4C]leucine from the culture medium by rabbit reticulocytes (Ueno et al. 1969). Several mycotoxins produced by Fusorium species were screened. Protein synthesis was inhibited by diacetoxyscirpenol, fusarenon-X and nivalenol when administered at very low concentrations (0.05-2,50 pg/ml). The cells were less sensitive to zearalenone (10.0 pg/ml). Other mycotoxins including aflatoxin B,, citrinin and butenolide had virtually no effect on protein synthesis at 50 pg/ml. Bioassays have also been developed as indicators for mycotoxins. In this role they cannot identify a toxin but they can point to its possible presence. Carnaghan et al. (1963) used ducklings to detect aflatoxin in animal feeds. Harwig & Scott (1971) found brine shrimps to be extremely sensitive to citrinin and patulin. Dobias et al. (1980) suggested Drosophila as a bioassay system for the antibiotic ramihyfin A.

Table 1. Some examples of the mycotoxin/cell culture combinations evaluated in cyto-

toxicity studies Mycotoxin

Alfatoxin B, Patulin Aflatoxin B, Ochratoxin A Aflatoxin B, Cyclochlorotine Luteosk yrin Nivalenol Patulin Penicillic acid Rubratoxin Fusarenon-X Diacetoxyscirpenol Zearalenone Patulin PR Toxin

Cell culture

Human embryonic lung Rat and mouse fibroblasts Monkey kidney epithelium

Reference Legator et al. 1965 Powell 1966 Engelbrecht & Purchase 1969

Rat liver Rat kidney Rat lung Human malignant epithelium cells i.e. HeLa cells

Umeda 1971

Mouse leucocytes

Nakano et ul. 1973

Pig, calf and turkey testes Rat and mouse fibroblasts Rat liver

Vanyi & Szailer 1974 Stott & Bullerman 1975

Auiard et af. 1979

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Bioassay methods for mycotoxin detection This antibiotic showed high insecticidal activity and low mammalian toxicity. Application of Cytotoxicity Tests to Screen Fungal Cultures

The rabbit reticulocyte test was applied in Japan to screen Fusarium isolates from cereals and animal feeds for their ability to produce toxic substances (Ueno et al. 1973). In this study the culture extracts were also intraperitoneally administered to mice and analysed for mycotoxins (trichothecenes) by thin layer chromatography. In the majority of cases where the extracts inhibited protein synthesis in the rabbit reticulocytes, to a marked degree, a corresponding toxic effect was observed in the mice. The effects observed included cellular abnormalities in the actively dividing cells in the thymus, bone marrow, small intestine, testes and ovary. These responses are characteristic of those when animals are challenged with trichothecenes. It is significant that chemical analysis of the culture extracts used in this experiment contained one or more of the following trichothecenesfusarenon-X, diacetoxyscirpenol, nivalenol, neosolaniol, HT-2 toxin and T-2 toxin. Other studies were also carried out in Japan using a cytotoxicity test to screen fungi isolated from food for toxic substances. They were undertaken during an investigation into the possible cause of a high mortality rate caused by cerebrovascular and liver diseases in the human population living in mountain and coastal villages. In these investigations fungal culture filtrates and mycelium extracts were applied to cultures of HeLa cells (This is a line of human malignant epithelium cells) and they were also injected subcutaneously into mice. The majority of the cultures toxic to the cells also produced an effect in the mice such as damage to the liver, kidneys and death (Saito et al. 1971, 1974). The predominant toxic fungi were Aspergillus ochraceus, A. versicolor, Pencillium citrinum and P . cyclopium. No work was done to identify the chemical nature of the toxic substances. Tracheal Organ Cultures

The value of a cytotoxicity test is enhanced if it is sensitive to a large number of known mycotoxins. The sensitivity of tracheal organ cultures

amount of mycotoxin detectable by tracheal organ cultures

Table 2. Minimum

Aflatoxin B , Aflatoxin B, Aflatoxin G , Gliotoxin

Ochratoxin Patulin Sporidesmin

Sporidesmin B Sterigmatocystin

0.200

13.67

0.89 0.85 1.75 6.42 1.39 1.63 0.12

* Lethal concentration (LC,,) of toxin in culture medium for a 50% response. prepared from day old chicks for 9 mycotoxins has been investigated (Cardeilhac et al. 1972). The cells lining the trachea possess hair-like projections, known as cilia, whose function is to ‘sweep’ mucous and foreign bodies out of the respiratory tract. The effect of mycotoxins on the movement of the cilia has been studied using tracheal rings maintained alive in a culture medium for 48 h. Mycotoxins differed considerably in the amount required to cause 100% loss of ciliary movement. Of those mycotoxins screened sterigmatocystin was the most potent, judged on the lowest dose to exert an effect. The minimum detectable amount of each mycotoxin assayed is set out in Table 2 Recent Progress Towards Developing Cytotoxicity Tests

For a cytotoxicity test to be of practical use it must be easy to perform and preferably possess a wide spectrum of sensitivity. The tests described so far have certain limitations. The rabbit reticulocyte test needs special facilities for handling radiolabelled compounds and the tracheal organ culture requires a readily available supply of day old chicks. Furthermore these tests differ in their sensitivity to various mycotoxins, e.g. aflatoxin B, has little effect on protein synthesis in rabbit reticulocytes though is able to suppress ciliary movement in tracheal organ cultures. The most convenient indication of cytotoxicity is cell death or some change in cell morphology easily recognized by microscopic

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examination. The work of Saito et al. (1971, 1974) applied microscopic examination to determine cytotoxicity but at that time fewer mycotoxins were known to exist than today. The increasing number of mycotoxins discovered in recent years makes some of the earlier studies inadequate in assessing the value of cytotoxicity tests in screening for all the known mycotoxins. One of the most recent and comprehensive studies investigated the effect of 33 mycotoxins on HEp-I1 and Chang cells (Robb & Norval 1983). Both these cell lines are of human epithelial type, Chang cells originate from the liver. The cells were cultured on glass coverslips in one ml of medium into which 1.0 jd of a standard mycotoxin solution was added. After 48 h incubation the cover slips were removed from the medium, fixed in ethanol, stained and microscopically examined. The most dilute solution of mycotoxin having any cytotoxic effects such as growth inhibition, cell rounding or detachment from the cover slip was recorded. Eight of the mycotoxins, namely aflatoxin G,, cytochalasin B,, luteoskyrin, moniliformin, ochratoxin A, rubratoxin and scopoletin failed to have any effect on either cell line. Those mycotoxins more commonly found to occur in stored products and which were toxic to both cell lines included aflatoxins B,, B, and G,, citrinin, sterigmatocystin and several trichothecenes. The Chang line tended to be the more sensitive and was able to detect as little as 0.01 ng of T-2 toxin. For the trichothecenes and sterigmatocystin the cytotoxicity test was more sensitive than thin layer chromatography (TLC), though for the aflatoxins, citrinin, ochratoxin A and zearalenone TLC was more sensitive. A more rapid cytotoxicity test dispensing with the need for microscopic examination of the cells has been devised using baby hamster kidney cells (BHK 21/C13), derived from oneday-old Syrian hamsters (Sanders 1984). This test was developed as a bioassay system for the detection of mycotoxins in animal feeds. It was designed to cope with a large number of samples and to eliminate or reduce subjective judgement when determining the end point. The bioassay is based on the cytotoxic effect of mycotoxins on cultured cells and is performed in 96 well microtitre plates. Twofold dilution of a mycotoxin standard or feed extract are made in the plate and cells are added to

each well. The metabolic activity of a relatively large number of cells in a small volume of medium, as in a microtitre plate well, rapidly lowers the pH. The medium contains phenol red as an indicator which, with pH fall, becomes yellow. Any wells not containing metabolically active cells remain at the original pH (7.2) and are red in colour. The end point of the bioassay is taken as the highest dilution of toxin at which the pH has not fallen. The sensitivity of this assay to 15 mycotoxins was evaluated. In common with the assay devised by Robb & Norval(l983) the BHK cells were most sensitive to the trichothecenes, e.g. T-2 toxin (2.0 ng) and diacetoxyscirpenol (5.0 ng), whereas it was relatively insensitive to other mycotoxins, e.g., aflatoxin B, (1200 ng), citrinin (2400 ng) and ochratoxin A (5000 ng). Overall, this bioassay was less sensitive than that of Robb & Norval (1983) which might be attributable to differences in the technique. Microscopic examination of the cells in wells where the pH had fallen did not reveal any cytotoxic effects. All wells where the pH had not fallen contained cells showing a wide range of cytotoxic effects. The Application of Cytotoxicity Tests in Mycotoxin Analysis There is a big step between determining the sensitivity of a tissue culture to pure mycotoxins and applying it to detect mycotoxins in naturally contaminated products. It is clearly essential that, in the absence of any mycotoxins, a concentrated extract of the product is not toxic to the cells. Problems of this kind have occurred with the brine shrimp larva (Artemia salina) bioassay (Harwig & Scott 1971) which is particularly sensitive to some trichothecenes (Eppley 1974). Unfortunately it is not suitable for screening certain products, such as silage and compound feedingstuffs if they contain longchain fatty acids because they are toxic to brine shrimps (Curtis et al. 1974). Further evaluation of this bioassay concluded that it was unsuitable as a biological screening system for the presence of mycotoxins in animal feedingstuffs because of the high incidence of false positive results in the absence of aflatoxin B,, ochratoxin A and T-2 toxin (Prior 1979). Cytotoxicity tests are less vulnerable to interfering substances because of their sensitivity to mycotoxins. This enables extracts to be diluted

Bioassay methods for mycotoxin detection sufliciently to overcome the effects of interfering substances yet still retain their toxicity if mycotoxins are present. However, they are not used widely for mycotoxin screening though their value has been demonstrated in investigating the cause of excessive activity, poor feathering and stunted growth affecting several broiler flocks in Scotland during the winter of 198C81 (Robb et al. 1982). Feed extracts were toxic to HEp I1 cells and subsequent chemical analysis of the feed detected both diacetoxyscirpenol and deoxynivalenol. This is a very good illustration of the value of cytotoxicity tests as a preliminary screen for some mycotoxins. Unlike many of the chemical analyses they do not require high capital cost equipment (e.g. GC, HPLC and mass spectrometers) and highly skilled operators. They cannot replace chemical methods because they are insufficiently selective to identify individual compounds. Their strength and main application lies in being able to screen products for biological activity and identify those samples for which a specific chemical analysis is justified. In this way they complement chemical analysis and may alert to the presence of a toxin even when its chemical identity is unknown.

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