Metabolism of N-nitroso-2,6-dimethylmorpholine by isozymes of rabbit liver microsomal cytochrome P-450

D. M. Kokkinakis, D. R. Koop, D. G. Scarpelli, Dennis Koop, P. F. Hollenberg

Research output: Contribution to journalArticle

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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-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.

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

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Cytochrome P-450 Enzyme System
Isoenzymes
Rabbits
Liver
Microsomes
Phenobarbital
Cricetinae
Antibodies
Liver Microsomes
N-nitroso-2,6-dimethylmorpholine
NADPH-Ferrihemoprotein Reductase
Mesocricetus
Xenobiotics
Hydroxylation
Mixed Function Oxygenases
Carcinogens
Phospholipids
Ethanol

ASJC Scopus subject areas

  • Cancer Research
  • Oncology

Cite this

Kokkinakis, D. M., Koop, D. R., Scarpelli, D. G., Koop, D., & Hollenberg, P. F. (1985). Metabolism of N-nitroso-2,6-dimethylmorpholine by isozymes of rabbit liver microsomal cytochrome P-450. Cancer Research, 45(2), 619-624.

Metabolism of N-nitroso-2,6-dimethylmorpholine by isozymes of rabbit liver microsomal cytochrome P-450. / Kokkinakis, D. M.; Koop, D. R.; Scarpelli, D. G.; Koop, Dennis; Hollenberg, P. F.

In: Cancer Research, Vol. 45, No. 2, 1985, p. 619-624.

Research output: Contribution to journalArticle

Kokkinakis, DM, Koop, DR, Scarpelli, DG, Koop, D & Hollenberg, PF 1985, 'Metabolism of N-nitroso-2,6-dimethylmorpholine by isozymes of rabbit liver microsomal cytochrome P-450', Cancer Research, vol. 45, no. 2, pp. 619-624.
Kokkinakis, D. M. ; Koop, D. R. ; Scarpelli, D. G. ; Koop, Dennis ; Hollenberg, P. F. / Metabolism of N-nitroso-2,6-dimethylmorpholine by isozymes of rabbit liver microsomal cytochrome P-450. In: Cancer Research. 1985 ; Vol. 45, No. 2. pp. 619-624.
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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-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.",
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AU - Hollenberg, P. F.

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N2 - 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.

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