Aflatoxin B1 (AFB1) is a potent toxin, mutagen, and one of only 30 substances described as a human carcinogen. The long term objective is to understand the mechanisms of carcinogenicity of this and related compounds so that rational approaches can be made toward elimination of their effects, if not their exposure, on human and animal health. The rainbow trout model has proven a useful comparative species with extremely high sensitivities which permit tumor studies of small quantities of scarce aflatoxins. The studies in this proposal will not only improve our understanding of aflatoxin toxicity, but also will help illucidate mechanisms underlying the high sensitivity of this species, and thus more clearly define the limits of its usefulness for studies related to human and environmental health. This proposal aims to describe the carcinogenicity of AFB1 and its major metabolites in trout (AFL, AFM1, AFL-M1) in terms of their cellular metabolism, detoxication, DNA adduct formation and persistence, and molecular details of epoxide-DNA interaction.
In specific aim 1, chemical and enzymatic syntheses will be used to produce tritiated and untritiated aflatoxins from available precursors. These 4 aflatoxins will be chemically activated with chloroperoxybenzoic acid to their 8,9-epoxides, reacted with DNA, and their major adducts described by HPLC behavior, UV spectroscopy and mass spectral analysis. These standards will be used to determine the type of adducts formed by each aflatoxin biologically. Each aflatoxin will be incubated in isolated hepatocytes, and its relative rates of production of DNA adducts, specific metabolites, and conjugates determined. The persistence of DNA adducts from each aflatoxin will be examined in vivo and in vitro. Each aflatoxin will be reacted with a 32p-end labeled sequenced 260bp DNA fragment, cleaved where adducts form, and the nucleotide specificity and neighboring sequence effects compared on DNA sequencing gels. The effects of varying aflatoxin-epoxide structure on intercalation and reactivity with DNA will be investigated.
In specific aim 2 the relative carcinogenicities and covalent binding indices of all 4 aflatoxins will be determined by embryo exposure. Very large sample sizes will provide precise tumor and DNA-binding dose response curves for AFB1 and AFL, by embryo exposure and juvenile feeding protocols. These studies will provide a comprehensive molecular description of the metabolism and initiation characteristics of AFB1 and its metabolites in trout.
