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Formation of Hydroxy Derivatives, Aldehydes, and Nitrite from N-Nitrosomethyl-n-amylamine by Rat Liver Microsomes and by Purified Cytochrome P-450 IIB1¹

Eppley Institute for Research in Cancer, University of Nebraska Medical Center, Omaha, Nebraska 68105 [C. J., S. S. M., J. N.], and Department of Biochemistry, University of Medicine and Dentistry-New Jersey Medical School, Newark, New Jersey 07103 [H. I., M. J. L., C. S. Y.]

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, K_m values for uninduced microsomes were, respectively, 1.6, 1.2, 0.3, and 1.1 mM, with V_max of 0.08, 0.26, 1.06, and 0.15 nmol/min/mg protein. With PB-induced microsomes, all 4 K_m 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.

¹ This work was supported by NIH Grants RO1-CA-35628 and RO1-CA-37037, Core Grant CA-36727 from the National Cancer Institute, and Core Grant ACS-SIG-16 from the American Cancer Society. Some of these results were presented at the 79th Annual Meeting of the. American Association for Cancer Research, May 25–28, 1988 (1).

² Visiting scientist from the Cancer Institute, Chinese Academy of Medical Sciences, Beijing, People's Republic of China.

³ To whom requests for reprints should be addressed.

⁴ Present address: Department of Chemical Biology and Pharmacognosy, College of Pharmacy, Rutgers University, Piscataway, NH 08855.

Received 3/16/89. Revised 6/19/89. Accepted 7/ 6/89.

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