Pyrrolizidine alkaloids (PAs) are hepatotoxic and carcinogenic alkaloids produced by Senecio and a number of other plant genera. Humans are exposed to PAs through ingestion of herbal teas, honey from bees or milk from cows or goats foraging on plants such as tansy ragwort (Senecio jacobaea). PAs undergo two major routes of metabolism, dehydrogenation to the toxic pyrrole derivative, dehydroretronecine (DHP), or N-oxidation which yields a detoxicated metabolite. These reactions have been ascribed to the cytochrome P-450-dependent mixed-function oxidase (mfo) system. Mammalian species vary widely in their susceptibility to PAs. Rats are quite susceptible, whereas, guinea pigs and rabbits are relatively resistant. Epidemiological evidence and accidental poisoning incidences strongly suggest that humans are very susceptible to PA-induced toxicity. The degree of susceptibility within a species, such as the rat, is strongly influenced by age, sex and previous exposure to environmental xenobiotics. The mechanism for such differences in sensitivity appears to be due to alterations in levels of the various monooxygenases. In this study, we will compare the metabolism of various PAs in vitro and in vivo in rat, rabbit and guinea pig. We will then examine the properties of different isozymes of cytochrome P-450 involved in DHP and N-oxide formation and investigate the existence, as well as the catalytic mechanism of formation, of other PA metabolites. In addition, the role of the less-studied flavin- containing monooxygenase (FMO) in PA N-oxiodation will be examined. These monooxygenases will be purified and their polyclonal antibodies utilized to assess the role of the individual enzymes in PA metabolism and to relate activity to changes in the amount of the protein (determined by immunoquantitation) as regulated by development and xenobiotic exposure. The activities and amounts of these monooxygenases will be related to the in vivo formation of covalent adducts of DNA, following administration of PAs. Human liver samples will also be analyzed for PA metabolism and the role played by individual monooxygenases studied, in order to compare humans to the animal models. These experiments are designed to ultimately provide us with an understanding of the genetic, developmental and environmental factors which determine the metabolic disposition of PAs and, hence, their toxicity. Such knowledge can be used to predict the relative risk of individuals and human populations to PAs.
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