Mycotoxins Aflatoxin

Hepatic carcinomas in rainbow trout grown in hatcheries have been linked to feed contaminated with aflatoxin. However, the current interest in aflatoxin is related to experimental carcinogenesis. Hendricks (1994) reviewed the carcinogenicity of aflatoxin in fish and other nonmammalian species.

Epizootics of hepatic carcinomas were discovered after dry feeds for trout came into wide use during the 1950s (Hueper and Payne, 1961; Rucker et al., 1961; Wood and Larson, 1961; Scarpelli et al., 1963), although earlier problems with hepatic neoplasms had occurred in hatchery-reared salmonids (Haddow and Blake, 1933; Nigrelli, 1954; Wales and Sinnhuber, 1966). Aflatoxin in cottonseed meal was the primary cause of these epizootics (Wolf and Jackson, 1963; Ashley et al., 1964; Halver, 1967; Sinnhuber, 1967); however, carcino-genicity of aflatoxin was enhanced by cyclopropenoid fatty acids (malvalic and sterculic acids) occurring naturally in cottonseeds (Lee et al., 1968, 1971; Sinnhuber et al., 1968, 1974; Hendricks et al., 1980a). Epizootics of hepatic carcinomas have occurred more recently (Majeed et al., 1984; Rasmussen et al., 1986), but problems in aquaculture have been reduced by avoiding feed ingredients with high concentrations of aflatoxin (Goldblatt, 1967). Feed ingredients most likely to be contaminated with aflatoxin are corn, cottonseed and peanuts (Lovell, 1989).

Aflatoxin is a mycotoxin produced by certain strains of Aspergillus flavus and Aspergillus parasiticus (Busby and Wogan, 1984). Several types of aflatoxin are produced by these fungi, but AFB1 is the major component and is also the form with the greatest potential carcinogenicity (Ayres et al., 1971). Aflatoxin B1 is not carcinogenic until conversion to the electrophilic 8,9-epoxide (2,3-oxide in older references), which can form adducts with DNA (Swenson et al., 1977; Baertschi et al., 1988). This metabolic activation is mediated by cytochrome P-450, and the extreme carcinogenicity of AFB1 in rainbow trout may be related to the preferential formation of the ultimate carcinogen rather than the formation of less carcinogenic metabolites (Williams and Buhler, 1983; Bailey et al., 1988). Aflatoxin B1 is also metabolized to compounds that can be conjugated and excreted; however, in rainbow trout some of these metabolites are carcinogenic, including aflatoxin Mi (Sinnhuber et al., 1974), aflatoxin Qi (Hendricks et al., 1980a), and aflatoxicol (Schoenhard et al., 1981). Aflatoxicol is a major metabolite of AFB1 in rainbow trout, and the tendency to form aflatoxicol, rather than less carcinogenic metabolites, during metabolism of AFB1 could contribute to the sensitivity of rainbow trout to AFB1 (Schoenhard et al., 1981).

Types of neoplasms in rainbow trout exposed to aflatoxin are hepatocellular adenomas, hepatocellular carcinomas, and mixed carcinomas containing both hepatocellular and cholangiolar components (Nunez et al., 1989, 1991). Hepatocellular adenomas (also termed basophilic nodules; Hendricks, 1982) consist of basophilic cells with less glycogen than normal hepatocytes. Hepatocytes within these adenomas are usually organized in tubules having the normal two-cell thickness. Compression and invasion of adjacent tissue are absent. Hepatocellular adenomas are uncommon and appear to be a transitional stage between preneoplastic basophilic foci and hepatocellular carcinoma (Hendricks et al., 1984b; Nunez et al., 1991). A trabecular pattern with well differentiated hepatocytes is the most common form of hepatocellular carcinoma (Hendricks et al., 1984b). These carcinomas are distinguished from hepatocellular adenoma by their invasiveness and expansion of trabeculae to five or more cells thick (Nunez et al., 1991). Metastases and emboli of carcinoma cells occur (Hueper and Payne, 1961; Wood and Larson, 1961; Ashley and Halver, 1963; Yasutake and Rucker, 1967; Nunez et al., 1989), but experimental studies are usually terminated before metastasis is observed.

Although mixed carcinomas are usually the most common neoplasm in rainbow trout exposed to aflatoxin, experimental exposures sometimes result in only hepatocellular carcinomas (Nunez et al., 1991). Hepatocytes within neoplasms caused by aflatoxin can function normally so that affected fish survive even after the liver has been almost totally replaced by neoplastic tissue (Hendricks, 1982).

An unusual lesion in rainbow trout fed aflatoxin is pancreatic acinar cell metaplasia within hepatocellular carcinomas (Hendricks et al., 1984b). Unlike many other teleosts, salmonids do not normally have pancreatic acini associated with the hepatic portal veins within the liver (Yasutake and Wales, 1983). Therefore, occurrence of exocrine pancreatic cells within the liver of aflatoxin-exposed rainbow trout is probably related to the origin of both tissues from a single pluripotential stem cell.

Fish species and strains vary dramatically regarding their sensitivity to aflatoxin. Rainbow trout are more sensitive to the carcinogenic action of dietary aflatoxin than are other animals studied (Busby and Wogan, 1984; Hendricks, 1994); 14% of the rainbow trout fed 0.4 mg AFB1 kg-1 of feed developed liver neoplasms after 15 months (Lee et al., 1968). Shasta strain rainbow trout are the most sensitive strain of rainbow trout (Sinnhuber et al., 1977; Bailey et al., 1989) and are the most commonly used fish in studies involving aflatoxin-induced carcinogenicity. However, this sensitivity is not a universal feature of fish or even of salmonids. Rats of the Fischer strain are more sensitive than coho salmon (Halver et al., 1969; Wogan et al., 1974; Bailey et al., 1988) or guppies (Poecilia reticulata) (Sato et al., 1973). Sockeye salmon (Oncorhynchus nerka) fed aflatoxin develop carcinomas only if synergists, such as cyclopropenoid fatty acids, are included in the diet (Wales and Sinnhuber, 1972). Not only is a high dose of AFB1 required for coho salmon to develop neoplasms, but the neoplasms that develop in coho salmon are adenomas rather than carcinomas. Compared with salmonids, channel catfish (Ictalurus punctatus) are much less sensitive to the acute and oncogenic properties of AFB1 (Ashley, 1970; Jantrarotai and Lovell, 1990; Jantrarotai et al., 1990). The low sensitivity of channel catfish could be related to incomplete absorption and rapid elimination of AFB1 (Plakas et al., 1991), but further study is needed to determine the mechanisms of protection from aflatoxin by this species.

Aflatoxin can also be used to initiate carcinogenesis before fish hatch. Rainbow trout embryos dipped in a solution of AFB1 for 30 min will develop hepatic neoplasms 9-12 months later (Sinnhuber and Wales, 1974; Wales et al., 1978; Hendricks et al., 1980d). Age of the exposed embryo is important because exposure after liver development increases sensitivity to AFB1 (Wales et al., 1978). An AFB1 concentration of 0.125 mg l-1 and a duration of exposure of 30 min resulted in an incidence of hepatic neoplasms of 5% for 9 months (Nunez et al, 1989).

Exposure of fish embryos or yolk-sac larvae can also be accomplished by microinjection of carcinogen, which offers the advantages of reducing the amount of carcinogen needed and ensuring exposure to water-insoluble compounds (Metcalf and Sonstegard, 1984; Black et al., 1985b; Metcalfe et al., 1988). Both rainbow trout and coho salmon have been used successfully for embryo injection of AFB1 (Black et al., 1988), and coho salmon offer the advantage of relatively large eggs (200 mg).

Other mycotoxins

Versicolorin A and sterigmatocystin are synthesized by Aspergillus spp. and are precursors in the synthesis of AFB1 by A. flavus and A. parasiticus. Both of these mycotoxins caused hepatic carcinomas in the rainbow trout embryo exposure assay (Hendricks et al., 1980b).

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