Chemically induced mammary gland tumorigenesis in the rat is the model system for studies of breast cancer. However, the mechanism(s) underlying this process by N-substituted aryl compounds has not been elucidated. Required for their carcinogenicity is oxidation to the N-hydroxy compounds which may then be activated by one electron oxidation (OEO) involving mammary gland peroxidase (MGP) to nitroxyl free radicals (NFR). Our proposed studies have 4 specific aims: 1) to establish whether susceptibilities to OEO of 4 isomeric N-fluorenylhydroxamic acids correlate with their tumorigenicities for the rat mammary gland: N-hydroxy-N-2-fluorenylacetamid (N-OH-2-FAA) Greater that N-OH-3-FAA is dominated by N-OH-1-FAA Greater than N-OH-4-FAA. It is anticipated that the less carcinogenic isomers will be oxidized to NFR, as are the carcinogenic isomers, but that the kinetic properties of each NFR will differ. Since these properties may determine if the NFR or its dismutation products, the nitrosofluorene and acetate ester of the hydroxamic acid, interact with cellular acceptors, rates of formation and dismutation of NFR and rates of product formation will be measured. Types of interactions with macromolecules with consideration of differing chemical reactivities of the isomeric products and specificities of acceptors in the susceptible (mammary gland) vs nonsusceptible (female rat liver) tissues will be determined; 2) to partially purify MGP and determine its subcellular localization, substrate specificity and estrogene-inducibility. If MGP is found to catalyze OEO of N-OH-2-(and -3-)FAA to NFR, the resulting interaction products with mammary gland nuclei (DNA adducts and acetylated chromatin) will be determined in vitro and in vivo. This will provide the most direct assessment of the role of MGP in activation of mammary gland carcinogens; 3) to determine whether rat mammary gland contains flavin-monooxygenase and can N-hydroxylate carcinogenic N-arylamines, and whether N-hydroxylamines undergo OEO by MGP to NFR. Since these NFR do not dismutate, their direct interactions with cellular acceptors will be investigated; and 4) to determine whether lactoperoxidase of lactating mammary gland or rat milk catalyzes OEO of N-OH-2-FAA to NFR, and to identify resultant interaction products with milk protein and lipid. These interactions may counteract the activation of N-OH-2-FAA by lactoperoxidase. These methodologies will be used: electron spin resonance and UV spectroscopy, gas and liquid chromatography, kinetic and computer analyses.
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