Formation of hydroxy derivatives, aldehydes, and nitrite from N-nitrosomethyl-n-amylamine by rat liver microsomes and by purified cytochrome P-450 IIB1

Abstract

The metabolism was examined of the esophageal carcinogen N-nitrosomethylamylamine (NMAA) by liver microsomes and slices from adult male Sprague-Dawley rats. Hydroxylation at C-2 to C-5 of the amyl group to give stable hydroxy-NMAAs was studied by gas chromatography-thermal energy analysis to determine the products. Microsomal metabolism produced mainly 4-hydroxy-NMAA, proceeded optimally in 100 mM phosphate at pH 7.4, and showed no sex differences. Induction by phenobarbital (PB) and 3-methylcholanthrene produced effects which were similar in slices and microsomes, with PB inducing hydroxylation at all positions and 3-methylcholanthrene specifically inducing 3-hydroxylation by factors of 2- and 6-fold. Clofibrate and isoniazid treatments did not affect NMAA metabolism by liver slices. Aroclor-1254 strongly induced microsomal 2- and 3-hydroxylation. For 2- to 5-hydroxylation, Km values for uninduced microsomes were, respectively, 1.6, 1.2, 0.3, and 1.1 mM, with Vmax of 0.08, 0.26, 1.06, and 0.15 nmol/min/mg protein. With PB-induced microsomes, all 4 Km values were 0.4-0.7 mM. Liver microsomal production of nitrite and aldehydes from NMAA was determined colormetrically or (for pentaldehyde) by high-pressure liquid chromatography of the 2,4-dinitrophenylhydrazone. Uninduced microsomes produced nitrite, formaldehyde and pentaldehyde from 0.6 mM NMAA at rates that were, respectively, 0.15, 0.72, and 1.15 times that for 4-hydroxylation. PB especially induced depentylation, whereas 3-methylcholanthrene induced depentylation and denitrosation, but suppressed demethylation. A reconstituted system containing cytochrome P-450 IIB1 gave metabolite ratios similar to those in PB-induced microsomes. The results account for most of the possible primary metabolites of NMAA and demonstrate the selectivity for metabolism at each position.