Metabolism of N-Nitroso-2,6-dimethylmorpholine by Isozymes of Rabbit Liver Microsomal Cytochrome P-450

Demetri M. Kokkinakis, Dante G. Scarpelli, Paul F. Hollenberg, Dennis R. Koop, Minor J. Coon

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27 Scopus citations

Abstract

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-hy-droxypropylX2-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-450LM, which is induced by pretreatment with phenobarbital, exhibited the highest activity for the metabolism of cis-NNDM. The V™ for the formation of HPOP was 1.78 nmoi/min/nmoi cytochrome POu, and the apparent Km was 360 /*M. Cytochrome P-450LMJ, 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 LM3a inhibited NNDM metabolism by only 7%. In microsomes from untreated rabbits, inhibition by anti-LM2 and anti-LM3a 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.

Original languageEnglish (US)
Pages (from-to)619-624
Number of pages6
JournalCancer Research
Volume45
Issue number2
StatePublished - Feb 1 1985

ASJC Scopus subject areas

  • Oncology
  • Cancer Research

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    Kokkinakis, D. M., Scarpelli, D. G., Hollenberg, P. F., Koop, D. R., & Coon, M. J. (1985). Metabolism of N-Nitroso-2,6-dimethylmorpholine by Isozymes of Rabbit Liver Microsomal Cytochrome P-450. Cancer Research, 45(2), 619-624.