Vanderslice R R, Boyd J A, Eling T E, Philpot R M
Cancer Res. 1985 Nov;45(11 Pt 2):5851-8.
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.
对从兔膀胱黏膜制备的微粒体部分进行分析，以检测与细胞色素P - 450依赖性单加氧酶系统相关的酶和活性。微粒体制剂中存在还原型烟酰胺腺嘌呤二核苷酸磷酸：细胞色素P - 450还原酶（315单位/毫克蛋白质）、还原型烟酰胺腺嘌呤二核苷酸：细胞色素b5还原酶（920单位/毫克蛋白质）、细胞色素P - 450（0.22纳摩尔/毫克蛋白质）和细胞色素b5（0.31纳摩尔/毫克蛋白质）。通过免疫化学方法检测到细胞色素P - 450的个体同工酶，即2、5和6型，但未检测到4型。用苯巴比妥或2,3,7,8 - 四氯二苯并 - p - 二恶英处理兔子，并未改变膀胱制剂中这些同工酶的浓度。在膀胱微粒体部分观察到单加氧酶活性（皮摩尔产物/分钟/蛋白质），苯丙胺N - 去甲基化（290）、7 - 乙氧基试卤灵O - 脱乙基化（29）、7 - 乙氧基香豆素O - 脱乙基化（28）、苯并(a)芘羟基化（10）和2 - 氨基芴羟基化（1400）。通过高效液相色谱法和闪烁计数法测定2 - 氨基芴的代谢；通过色谱保留时间、紫外 - 可见光谱和质谱鉴定出两种产物，即2 - 亚硝基芴和2,2'-偶氮双芴。另外两种代谢产物初步鉴定为N - 羟基 - 2 - 氨基芴和一种环羟基化产物。2 - 氨基芴的代谢受到细胞色素P - 450 5型抗体或还原型烟酰胺腺嘌呤二核苷酸磷酸：细胞色素P - 450还原酶抗体以及一氧化碳（CO:O2，4:1）的抑制，但不受细胞色素P - 450 2型抗体的抑制。还观察到在对氧磷或氟化钠敏感的酶介导下，2 - 氨基芴在乙酸乙酯存在下的乙酰化作用（以及2 - 乙酰氨基芴的脱乙酰化作用）。
