title: Metabolism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) by cytochrome P450IIB1 in a reconstituted system.
abstract: Several previous studies have suggested that cytochrome P450IIB1 is involved in the bioactivation of the tobacco-specific carcinogen, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), in rats as well as in mouse lung microsomes. The present investigation was undertaken to study the metabolism of NNK by purified cytochrome P450IIB1 in a reconstituted system. The metabolites 4-hydroxy-4-(3-pyridyl) butyric acid (hydroxy acid), 4-oxo-4-(3-pyridyl) butyric acid (keto acid), 4-oxo-4-(3-pyridyl) butanol (keto aldehyde), 4-(methylnitrosamino)-1-(3-pyridyl-N-oxide)-1-butanone (NNK-N-oxide) and 4-oxo-4-(3-pyridyl)-1-butanol (keto alcohol) were quantitated by HPLC. The results showed that, in addition to alpha-hydroxylations, cytochrome P450IIB1 also catalyzed the formation of NNK-N-oxide efficiently, and to a certain extent, the conversion of NNK primary hydroxylation metabolites (keto aldehyde and keto alcohol) to secondary metabolites (keto acid and hydroxy acid). Cytochrome b5 at a ratio of 1:1 or 2:1 to P450IIB1 had no significant effect on the metabolic activities and profiles of NNK. The apparent Km values for the formation of keto aldehyde, NNK-N-oxide and keto alcohol were respectively 191.2, 131.4 and 318.0 microM with corresponding apparent Vmax values of 89.7, 295.5 and 333.3 pmol/min/nmol P450, indicating that hydroxylation at the alpha-methyl position is preferred over the alpha-methylene position. Measurement of formaldehyde, a product derived from the alpha-methyl hydroxylation, was developed as a convenient method to study NNK metabolism. Thiourea activated cytochrome P450IIB1-catalyzed NNK metabolism significantly. Phenethyl isothiocyanate, an inhibitor of NNK-induced lung carcinogenesis, inhibited P450IIB1-catalyzed NNK demethylation in a concentration-dependent manner. This work demonstrates that purified P450IIB1 can catalyze the conversion of NNK to most of its oxidative metabolites.
authors.affiliation: Laboratory for Cancer Research, College of Pharmacy, Rutgers University Piscataway, NJ 08855-0789.
authors.forename: Z
authors.initials: Z
authors.lastname: Guo
authors.forename: T J
authors.initials: TJ
authors.lastname: Smith
authors.forename: H
authors.initials: H
authors.lastname: Ishizaki
authors.forename: C S
authors.initials: CS
authors.lastname: Yang
mesh_terms: D000818:Animals; D002273:Carcinogens / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D019362:Cytochrome P-450 CYP2B1; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D017879:Isothiocyanates; D007700:Kinetics; D008745:Methylation; D008862:Microsomes, Liver / Q000201:enzymology; D009602:Nitrosamines / Q000378:metabolism*; D010088:Oxidoreductases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051381:Rats; D013861:Thiocyanates / Q000494:pharmacology; D013890:Thiourea / Q000494:pharmacology
chemical_list: D002273:Carcinogens; D065607:Cytochrome P-450 Enzyme Inhibitors; D017879:Isothiocyanates; D009602:Nitrosamines; D013861:Thiocyanates; C058305:phenethyl isothiocyanate; C016583:4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; D019362:Cytochrome P-450 CYP2B1; D013890:Thiourea