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Bovine Bladder Mucosa Microsomal Cytochrome P-450 and 4-Aminobiphenyl N-Hydroxylase Activity¹

Department of Pharmacology, University of Miami School of Medicine, Miami, Florida 33101

The potential for metabolic activation of bladder carcinogens by the bladder mucosa was examined by determining if bladder mucosa microsomes contain cytochrome P-450, exhibit typical microsomal-substrate interactions, and are capable of mediating the N-hydroxylation of the bladder carcinogen, 4-aminobiphenyl (4-ABP). The carbon monoxide difference spectrum of reduced bovine bladder microsomes exhibited an absorption maximum at 450 nm and an absorption mininum at 405 nm, characteristic of cytochrome P-450. Bladder mucosa microsomes contained 0.13 nmol cytochrome P-450 per mg microsomal protein. Addition of aniline to bladder mucosa microsomes yielded a typical type 2 binding spectrum with a _max at 432 nm and a _min at 390 to 410 nm, identical to that observed with rat liver microsomes. Addition of the bladder carcinogen 4-ABP to either liver or bladder microsomes resulted in an atypical type 2 difference spectrum with a _max at 434 nm, a _min at 420 nm, and a steep increase in absorption between 420 and 370 nm. Compounds such as hexobarbital and 2-diethylaminoethyl-2, 2-diphenylvalerate hydrochloride, which exhibit type 1 spectra with rat liver microsomes, produced weak interactions with bladder mucosa microsomes with a small _min at 425 nm and no definitive _max at lower wavelength. Bovine bladder microsomes mediated reduced nicotinamide adenine dinucleotide phosphate-dependent N-hydroxylation of 4-ABP at a rate of 0.3 µmol N-hydroxy-4-aminobiphenyl per nmol cytochrome P-450 per 10-min incubation, 20 times the rate of 4-ABP N-hydroxylation observed with rat liver microsomes. No detectable N-hydroxylase activity was found with bovine liver microsomes. The rate of bladder mucosa microsomal-mediated N-hydroxylation was linear with respect to the concentration of cytochrome P-450 up to 2.44 nmol/ml. Bladder microsomal 4-ABP N-hydroxylase activity was partially inhibited by 2-[(2,4-dichloro-6-phenyl)phenoxy]ethylamine, implying that this activity is at least partially mediated by cytochrome P-450. These observations suggest that bladder carcinogens may be activated within the target tissue itself, the bladder mucosa, providing an alternative to liver metabolism as a mechanism for the activation of bladder precarcinogens.

¹ This research was supported by funds from USPHS Grant CA 14927 from the National Cancer Institute through the National Bladder Cancer Project.

² To whom requests for reprints should be addressed.

Received 7/21/80. Accepted 1/12/81.