Pyrrolizidine alkaloids (PAs) are plant derived toxins that have been shown to contaminate human food sources (herbal teas, honey, milk, cereals and grains). This contamination may be highly significant since PAs are responsible for numerous syndromes and are proven mutagens and carcinogens. The proposed experiments will isolate and identify the in vitro hepatic PA metabolites from the macrocyclic PAs retrorsine, seneciphylline and senecionine (S. vulgaris). In vitro kinetic studies using radiolabelled PAs will be performed to determine the rate of formation, interaction and disappearance of these PA metabolites. Upon identification of the PA metabolites, the in vitro experiments will be scaled up or the metabolites synthesized to provide material to test for in vitro toxicity. Isolated hepatocytes will be used to closely examine the mechanisms and nature of the hepatotoxicity exerted by the PAs and their metabolites. These isolated hepatocytes studies will involve both cytotoxicity and DNA repair. Protein PA adducts will be isolated using an in vitro hepatic microsomal incubation. Cysteine and glutathione will be used as model compounds in the incubation mixture to facilitate these experiments. These in vitro studies have been undertaken to elucidate the fundamental mechanisms regarding the mode of PA hepatotoxicity and will be extremely useful in future in vivo experiments.
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