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The role of cytochromes P-450 and flavin-containing monooxygenase in the metabolism of (S)-nicotine by rabbit lung.
Drug Metab Dispos. 1990 Jul-Aug; 18(4):418-28.DM

Abstract

Rabbit lung microsomes metabolize (S)-nicotine primarily to (S)-nicotine delta 1',5'-iminium ion, which is the precursor of (S)-cotinine, the major urinary metabolite of (S)-nicotine in mammals. (S)-Nicotine-N'-oxide and normicotine are also produced as minor metabolites. alpha-Methylbenzylaminobenzotriazole, a mechanism-based suicide inhibitor of rabbit lung cytochromes P-450 2 and 6, inhibited (S)-nicotine oxidation in parallel with inhibition of benzphetamine N-demethylation and ethoxyresorufin O-deethylation. Pretreatment of rabbits with TCDD or Aroclor 1260 had no effect and markedly inhibited (S)-nicotine oxidation, respectively, strongly suggesting that alpha-methylbenzylaminobenzotriazole inhibition was due to inactivation of rabbit lung P-450 2. Reconstitution with cytochromes P-450 2 and 5 demonstrated that only P-450 2 was active toward (S)-nicotine, yielding predominantly the iminium ion, with smaller amounts of nornicotine, (S)-nicotine N'-oxide, and an unknown metabolite also detected. The purified rabbit lung P-450 2-catalyzed oxidation of (S)-nicotine to (S)-nicotine delta 1',5'-iminium ion exhibited a Km of 70 microM and a Vmax of 1.5 min. Covalent binding of (S)-5-3H-nicotine to rabbit lung macromolecules was dependent upon rabbit lung P-450 2-catalyzed formation of the iminium ion. Antibodies raised against P-450 2 inhibited the rabbit lung microsomal metabolism of (S)-nicotine to (S)-nicotine delta 1',5'-iminium ion by almost 95%. Titration of reconstituted P-450 2 with cytochrome b5 produced a concentration-dependent inhibition of nicotine oxidase activity. Increasing the ratio of NADH to NADPH in incubations containing lung microsomes and (S)-nicotine decreased the yield of the iminium ion, confirming the inhibitory effect of cytochrome b5 on the P-450 2-catalyzed alpha-carbon oxidation reaction. NADH alone did not support the lung microsomal metabolism of (S)-nicotine. N'-oxidation of (S]-nicotine is catalyzed by purified pig liver flavin-containing monooxygenase. A number of experiments involving the use of P-450 inhibitors, titration with NADPH-cytochrome P-450 reductase antibodies, and determination of the pH-enzyme activity profile suggested that rabbit lung flavin-containing monooxygenase contributes to a small amount of the N'-oxide produced by rabbit lung microsomes. Further examination with purified flavin-containing monooxygenase isolated from rabbit lung microsomes demonstrated that (S)-nicotine is a poor substrate for this enzyme. The low yield of N'-oxide, relative to other metabolites, in rabbit lung is uncharacteristic for most mammalian tissues and presumably reflects the unusual substrate specificity of rabbit lung flavin-containing monooxygenase.

Authors+Show Affiliations

Department of Food Science and Technology, Oregon State University, Corvaltis 97331.No affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

1976062

Citation

Williams, D E., et al. "The Role of Cytochromes P-450 and Flavin-containing Monooxygenase in the Metabolism of (S)-nicotine By Rabbit Lung." Drug Metabolism and Disposition: the Biological Fate of Chemicals, vol. 18, no. 4, 1990, pp. 418-28.
Williams DE, Shigenaga MK, Castagnoli N. The role of cytochromes P-450 and flavin-containing monooxygenase in the metabolism of (S)-nicotine by rabbit lung. Drug Metab Dispos. 1990;18(4):418-28.
Williams, D. E., Shigenaga, M. K., & Castagnoli, N. (1990). The role of cytochromes P-450 and flavin-containing monooxygenase in the metabolism of (S)-nicotine by rabbit lung. Drug Metabolism and Disposition: the Biological Fate of Chemicals, 18(4), 418-28.
Williams DE, Shigenaga MK, Castagnoli N. The Role of Cytochromes P-450 and Flavin-containing Monooxygenase in the Metabolism of (S)-nicotine By Rabbit Lung. Drug Metab Dispos. 1990 Jul-Aug;18(4):418-28. PubMed PMID: 1976062.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - The role of cytochromes P-450 and flavin-containing monooxygenase in the metabolism of (S)-nicotine by rabbit lung. AU - Williams,D E, AU - Shigenaga,M K, AU - Castagnoli,N,Jr PY - 1990/7/11/pubmed PY - 2001/3/28/medline PY - 1990/7/11/entrez SP - 418 EP - 28 JF - Drug metabolism and disposition: the biological fate of chemicals JO - Drug Metab Dispos VL - 18 IS - 4 N2 - Rabbit lung microsomes metabolize (S)-nicotine primarily to (S)-nicotine delta 1',5'-iminium ion, which is the precursor of (S)-cotinine, the major urinary metabolite of (S)-nicotine in mammals. (S)-Nicotine-N'-oxide and normicotine are also produced as minor metabolites. alpha-Methylbenzylaminobenzotriazole, a mechanism-based suicide inhibitor of rabbit lung cytochromes P-450 2 and 6, inhibited (S)-nicotine oxidation in parallel with inhibition of benzphetamine N-demethylation and ethoxyresorufin O-deethylation. Pretreatment of rabbits with TCDD or Aroclor 1260 had no effect and markedly inhibited (S)-nicotine oxidation, respectively, strongly suggesting that alpha-methylbenzylaminobenzotriazole inhibition was due to inactivation of rabbit lung P-450 2. Reconstitution with cytochromes P-450 2 and 5 demonstrated that only P-450 2 was active toward (S)-nicotine, yielding predominantly the iminium ion, with smaller amounts of nornicotine, (S)-nicotine N'-oxide, and an unknown metabolite also detected. The purified rabbit lung P-450 2-catalyzed oxidation of (S)-nicotine to (S)-nicotine delta 1',5'-iminium ion exhibited a Km of 70 microM and a Vmax of 1.5 min. Covalent binding of (S)-5-3H-nicotine to rabbit lung macromolecules was dependent upon rabbit lung P-450 2-catalyzed formation of the iminium ion. Antibodies raised against P-450 2 inhibited the rabbit lung microsomal metabolism of (S)-nicotine to (S)-nicotine delta 1',5'-iminium ion by almost 95%. Titration of reconstituted P-450 2 with cytochrome b5 produced a concentration-dependent inhibition of nicotine oxidase activity. Increasing the ratio of NADH to NADPH in incubations containing lung microsomes and (S)-nicotine decreased the yield of the iminium ion, confirming the inhibitory effect of cytochrome b5 on the P-450 2-catalyzed alpha-carbon oxidation reaction. NADH alone did not support the lung microsomal metabolism of (S)-nicotine. N'-oxidation of (S]-nicotine is catalyzed by purified pig liver flavin-containing monooxygenase. A number of experiments involving the use of P-450 inhibitors, titration with NADPH-cytochrome P-450 reductase antibodies, and determination of the pH-enzyme activity profile suggested that rabbit lung flavin-containing monooxygenase contributes to a small amount of the N'-oxide produced by rabbit lung microsomes. Further examination with purified flavin-containing monooxygenase isolated from rabbit lung microsomes demonstrated that (S)-nicotine is a poor substrate for this enzyme. The low yield of N'-oxide, relative to other metabolites, in rabbit lung is uncharacteristic for most mammalian tissues and presumably reflects the unusual substrate specificity of rabbit lung flavin-containing monooxygenase. SN - 0090-9556 UR - https://www.unboundmedicine.com/medline/citation/1976062/The_role_of_cytochromes_P_450_and_flavin_containing_monooxygenase_in_the_metabolism_of__S__nicotine_by_rabbit_lung_ L2 - http://dmd.aspetjournals.org/cgi/pmidlookup?view=long&pmid=1976062 DB - PRIME DP - Unbound Medicine ER -