Mammary gland (MG) carcinogenesis in the rat, chemically induced, has many similarities to human breast cancer. N- Arylhydroxamic acids induced MG tumors (epithelial and stromal) in female rats after systemic or direct application and sarcomas at injection sites. These carcinogens require activation to form covalent DNA adducts considered critical for initiation. One possible route to activation may be via peroxidases, intracellular and leukocytic. These peroxidases catalyze le-oxidation and/or halide-dependent oxidative cleavage or N-hydroxy-N-2- fluorenylacetamide (N-OH-2-FAA) to yield N-acetoxy-2-FAA and/or 2-nitrosofluorene (2-NOF), both of which are carcinogenic. 2-NOF is also an extremely potent direct mutagen. Both products, N-AcO-2-FAA directly and 2-NOF indirectly, react covalently with DNA. We propose to continue our investigation of peroxidative activation of N-fluorenyl-hydroxamic acids under the following specific aims: 1) To further characterize haloperoxidase-mediated oxidations of N-OH-2-FAA, and related carcinogens N-OH-3-FAA and N-hydroxy-N-2-fluorenylbenzamide (N-OH-2-FBA) using myeloperoxidase to investigate Br- vs Cl- specificity and lactoperoxidase to investigate I--dependent oxidations vs ring iodinations; 2) To further investigate the oxidation of N-OH-2-FAA by ubiquitous prostaglandin hydroperoxide synthase; 3) To increase peroxidative activity in the rat MG to facilitate metabolic studies; 4) To concurrently determine N,O-acyl-transferase activity in the rat MG since it may contribute to activation of N-OH-2-FAA; 5) To further increase the specific activity of MG peroxidase via chromatographic purification; 6) To characterize the partially purified MG peroxidase with respect to substrate, inhibitor and halide specificities, and by comparison to uterine and eosinophil peroxidase; 7) To determine if nucleic acid adducts are formed from N-fluorenylhydroxamic acids peroxidative conditions of le- oxidation and halide-dependent oxidative cleavage at the intermediate or product stage; 8) To determine the peroxidative activities in the rat peritoneal fluid and serosa; and to determine in vivo metabolites of the carcinogens N-OH-2-FAA and N-OH-2- FBA in peritoneal fluid. The chief methodologies to be used in the proposed studies ar HPLC, FPLC, UV spectrometry, affinity chromatography, and gel electrophoresis.
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