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Chemical or enzymatic oxidations of the carcinogen N-hydroxy-N-(2- fluorenyl)benzamide (N-OH-2-FBA) were investigated under the conditions facilitating one-electron oxidation or oxidative cleavage of N-hydroxy-N-(2-fluorenyl)acetamide (N-OH-2-FAA). HPLC methods were developed for separation and quantitation of the above hydroxamic acids and their respective oxidation products. To identify the products of oxidation of N-OH-2-FBA, N-(benzoyloxy)-2-FBA (N-BzO-2-FBA) was synthesized and shown to undergo ortho rearrangement to 1- and 3-BzO-2-FBA. Oxidation of N-OH-2-FBA (4.88 mM) with alkaline K3Fe(CN)6 in benzene was complete and yielded equimolar amounts of 2-nitrosofluorene (2-NOF) and the ester (chiefly N-BzO-2-FBA), indicative of one-electron oxidation to nitroxyl free radical which undergoes bimolecular dismutation. However, one-electron oxidation of N-OH-2-FBA (30 or 10 microM) by horseradish peroxidase/H2O2 at pH 7 or myeloperoxidase/H2O2 at pH 6.5 yielded only approximately 10% as much product as N-OH-2-FAA (30 microM). The addition of 0.1 mM Br- +/- 0.1 M Cl- at pH 4 to 6.5 increased 2-NOF formation in MPO/H2O2-catalyzed oxidations. Simulations of these oxidations with HOCl/Cl- or HOBr/Br- showed that the latter was more efficient, converting N-OH-2-FAA almost completely and less than or equal to 62% of N-OH-2-FBA to 2-NOF. The amounts of the ester (N- and o-BzO-2-FBA), which by itself did not contribute to 2-NOF formation or significant substrate regeneration, indicated that approximately 10% of 2-NOF originated from one-electron oxidation.(ABSTRACT TRUNCATED AT 250 WORDS)

Lactoperoxidase-catalyzed metabolism of N-hydroxy-N-2-fluorenylacetamide (N-OH-2-FAA) may be via one-electron oxidation to nitroxyl free radical which dismutates to equimolar N-acetoxy-N-2-fluorenylacetamide and 2-nitrosofluorene (2-NOF) and/or a Br(-)-dependent oxidative cleavage to 2-NOF. Hence, the 2-NOF:N-acetoxy-N-2-fluorenylacetamide ratios reflect the relative contributions of the two peroxidative pathways to the metabolism of N-OH-2-FAA. Peroxidative activities of rat uterus (UT) and mammary gland (MG) were extracted with a cationic detergent, cetyltrimethylammonium bromide (Cetab). MG extracts had 1 to 5% the specific activity of UT extracts when assayed with guaiacol as hydrogen donor. At 0.004% Cetab, which in the incubation media corresponds to the approximate physiological levels of 0.1 mM Br(-), oxidation of N-OH-2-FAA by UT extracts yielded a product ratio indicative of both peroxidative pathways with Br(-)-dependent oxidation prevailing. At 0.4% Cetab, one-electron oxidation was negligible and Br(-)-dependent conversion of N-OH-2-FAA to 2-NOF was markedly enhanced. At both 0.004 and 0.4% Cetab, MG extracts yielded only 2-NOF, suggesting solely Br(-)-dependent oxidation. With equivalent guaiacol units of peroxidative activities, MG extracts produced much lower amounts of 2-NOF than did UT extracts. The low specific activities of MG extracts necessitated the use of larger amounts of protein, which might have interfered with peroxidative metabolism. At 0.004% Cetab, formation of 2-NOF by the Br(-)-dependent pathway was greater at pH 5.5 than at 7.4. At acid pH, small amounts of 2-nitrofluorene were also formed by UT and MG extracts and could be attributed to further oxidation of 2-NOF. Peroxidative activities of the UT and MG extracts may be of granular leukocyte origin and their potential role in carcinogen activation and tumorigenesis is discussed.