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

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 mediat ing 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 Xma„ at 432 nm and a \mln 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 A™,, at 420 nm, and a steep increase in absorption between 420 and 370 nm. Compounds such as hexobarbital and 2diethylaminoethyl-2, 2-diphenylvalerate hydrochloride, which exhibit type 1 spectra with rat liver microsomes, produced weak interactions with bladder mucosa microsomes with a small A,™,, at 425 nm and no definitive Ama,< at lower wavelength. Bovine bladder microsomes mediated reduced nicotinamide adenine dinucleotide phosphate-dependent A/-hydroxylation of 4-ABP at a rate of 0.3 /imol /V-hydroxy-4-aminobiphenyl per nmol cytochrome P-450 per 10-min incubation, 20 times the rate of 4-ABP A/-hydroxylation observed with rat liver micro somes. No detectable A/-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. Blad der microsomal 4-ABP /V-hydroxylase activity was partially inhibited by 2-[(2,4-dichloro-6-phenyl)phenoxy]ethylamine, im plying that this activity is at least partially mediated by cyto chrome P-450. These observations suggest that bladder car cinogens 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.