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Metabolism of N-Nitroso-2,6-dimethylmorpholine by Isozymes of Rabbit Liver Microsomal Cytochrome P-450¹

Departments of Pathology and Molecular Biology and the Cancer Center, Northwestern University Medical School, Chicago, Illinois 60611 [D. M. K., D. G. S., P. F. H.], and Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109 [D. R. K., M. J. C.]

The cis isomer of N-nitroso-2,6-dimethylmorpholine (NNDM), a pancreatic carcinogen for the Syrian golden hamster, is metabolized by hamster liver microsomes to yield N-nitroso(2-hydroxypropyl)(2-oxopropyl)amine (HPOP) as the major product. Rabbit liver microsomes catalyze the metabolism of cis-NNDM to HPOP at a rate slower than that observed with hamster microsomes, but significantly faster than that obtained with rat microsomes. Pretreatment of rabbits with phenobarbital results in a 6-fold increase in the cis-NNDM hydroxylase activity of the rabbit microsomes to levels equal to that observed with the hamster; pretreatment of rabbits with other xenobiotics had no effect on the hydroxylation of cis-NNDM. The role of rabbit liver microsomal cytochrome P-450 in the metabolism of the cis isomer of NNDM was studied in the reconstituted system consisting of NADPH:cytochrome P-450 reductase, phospholipid, and cytochrome P-450. Cytochrome P-450_LM2, which is induced by pretreatment with phenobarbital, exhibited the highest activity for the metabolism of cis-NNDM. The V_max for the formation of HPOP was 1.78 nmol/min/nmol cytochrome P-450_LM2, and the apparent K_m was 360 µM. Cytochrome P-450_LM3a also catalyzed the metabolism of NNDM to HPOP at a significant rate (0.25 nmol/min/nmol cytochrome P-450). Of the four other isozymes of cytochrome P-450 (forms 3b, 3c, 4, and 6) tested in the reconstituted system, only forms 3b and 3c exhibited measurable activities (approximately 0.04 nmol of HPOP formed/min/nmol cytochrome P-450).

The addition of antibodies to isozyme 2 to microsomes from phenobarbital-treated rabbits resulted in approximately 95% inhibition of the metabolism of NNDM, while the addition of antibodies to LM_3a inhibited NNDM metabolism by only 7%. In microsomes from untreated rabbits, inhibition by anti-LM₂ and anti-LM_3a antibodies was 50 and 64%, respectively. The addition of antibodies to isozyme 3a to microsomes isolated from ethanol-treated rabbits caused approximately 90% inhibition of the metabolism of NNDM.

These data conclusively demonstrate that several forms of cytochrome P-450 can catalyze the metabolism of cis-NNDM and that isozymes 2 and 3a play important roles in the rabbit hepatic metabolism of NNDM to HPOP, the proximate carcinogenic metabolite.

¹ This work was supported in part by the Edith Patterson and Marie A. Fleming Cancer Research Funds; Northwestern University; and Grants CA 16954 and CA 34051 from the National Cancer Institute, USPHS.

² Recipient of National Institute on Alcohol Abuse and Alcoholism Grant AA06221.

³ To whom requests for reprints should be addressed.

Received 5/21/84. Accepted 11/ 6/84.