The cytochrome P-450 monooxygenase system of rabbit bladder mucosa: enzyme components and isozyme 5-dependent metabolism of 2-aminofluorene.

The microsomal fraction prepared from the mucosa of rabbit bladder was analyzed for the presence of enzymes and activities associated with the cytochrome P-450-dependent monooxygenase system. Reduced nicotinamide adenine dinucleotide phosphate:cytochrome P-450 reductase (315 units/mg protein), reduced nicotinamide adenine dinucleotide:cytochrome b5 reductase (920 units/mg protein), cytochrome P-450 (0.22 nmol/mg protein), and cytochrome b5 (0.31 nmol/mg protein) were present in the microsomal preparation. Individual isozymes of cytochrome P-450, forms 2, 5, and 6, but not form 4, were detected by immunochemical methods. Treatment of rabbits with either phenobarbital or 2,3,7,8-tetrachlorodibenzo-p-dioxin did not alter the concentrations of these isozymes in the bladder preparation. Monooxygenase activities (pmol product/min/protein) in the bladder microsomal fraction were observed for benzphetamine N-demethylation (290), 7-ethoxyresorufin O-deethylation (29), 7-ethoxycoumarin O-deethylation (28), benzo(a)pyrene hydroxylation (10), and 2-aminofluorene hydroxylation (1400). The metabolism of 2-aminofluorene was determined by high performance liquid chromatography and scintillation counting; two products, 2-nitrosofluorene and 2,2'-azoxybisfluorene, were identified by chromatographic retention times, ultraviolet-visible spectroscopy, and mass spectrometry. Two additional metabolites were tentatively identified as N-hydroxy-2-aminofluorene and a ring-hydroxylated product. The metabolism of 2-aminofluorene was inhibited by antibodies to cytochrome P-450 form 5 or to reduced nicotinamide adenine dinucleotide phosphate:cytochrome P-450 reductase and by carbon monoxide (CO:O2, 4:1), but not by antibodies to cytochrome P-450 form 2. Acetylation of 2-aminofluorene in the presence of ethyl acetate (and deacetylation of 2-acetylaminofluorene) mediated by an enzyme sensitive to inhibition by either paraoxon or sodium fluoride was also observed.