The overall objectives of our studies on cyclophosphamide (CP) are to: (a) obtain a detailed understanding of the metabolism of CP and the interaction of CP metabolites with tissue macromolecules, especially DNA, RNA and proteins; (b) elucidate the chemical and biochemical basis of CP toxicity arising as a consequence of interaction between activated CP metabolites and tissue macromolecules; and (c) apply information derived in the above studies to develop biochemical interventions for obviating (ameliorating) some of the undesirable toxicities of CP with little interference of its chemotherapeutic activity. Metabolism of [3H-chloroethyl]-CP(3H-CP) and [14C-CP]-CP (14C-CP) to macromolecular-binding metabolites and to cytotoxic and other metabolites, formed by purified cytochrome P-450, hepatic nuclear fractions and prostaglandin synthetase, will be investigated in vitro. New metabolites, and metagolite-macromolecular adducts will be identified, isolated and characterized. In vivo formation of these adducts and their role in organ specific toxicities, especially liver and lung toxicity, of CP and its activated analogs (ASTZ-Z-7557 and PS-CP) will be investigated. Studies will be undertaken to define relationships between specific biochemical alterations (lipid peroxidation, glutathione depletion, inhibition of antioxidant defenses, DNA damage, and formation and persistance of CP metabolite - macromolecular adducts) induced by CP and certain specific toxicities of CP, such as liver and lung toxicity. Much of the proposed work will be performed: (a) in vitro with 14C-CP, 3H-CP and some labeled metabolites of CP (e.g. 4-hydroxy-CP, didechlorodihydroxy-CP, phosphoramide mustard), nucleic acids (DNA, RNA, purine and pyrimidine-nucleoside polymers) and enzyme preparations (cytochromes P-450, prostaglandin synthetase and hepatic nuclei) from rats and mice; (b) in vivo with labeled-CP in mice and rats to determine the in vivo macromolecular binding of CP and its relation to toxicity; and (c) in vivo in rats and mice to elucidate the mechanism by which CP produces specific biochemical effects, e.g. glutathione depletion and lipid peroxidation. Particular methods to be used include: radiochemical techniques, thin layer chromatography, high pressure liquid chromatography, column chromatography, organic analytical techniques, and other chemical, biochemical and enzymological methods.
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