The role of cytochromes P-450 and flavin-containing monooxygenase in the metabolism of (S)-nicotine by rabbit lung.

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.