Matabolism 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 tobaccospecific carcinogen, 4-(methylnitrosamino)-1-(3-pyridy1)-1-butanone (NNK), in rat 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 4hydroxy4(3-pyridyl) butyric acid (hydroxy acid), 4-oxo4-(3-pyridyl) butyric acid (keto acid), 4-oxo4-(3-pyridyl) butanol (keto aldehyde), 4-(methylnitrosamino)-1-(3-pyridyl-N-oxide)-1-butanone (NNK-N-oxide) and 4-oxo-4-(3-pyridy1)-1-butanol (keto alcohol) were quantitated by HPLC. The results showed that, in addition to a-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:l or 2:l 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 μM with corresponding apparent Vmax values of 89.7, 295.5 and 333.3 pmol/min/nmol P450, indicating that hydroxylation at the α-methyl position is preferred over the α-methylene position. Measurement of formaldehyde, a product derived from the α-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 NNKinduced 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.