Interaction of constitutive and phenobarbital-induced cytochrome P-450 isozymes during the sequential oxidation of benzphetamine. Explanation for the difference in benzphetamine-induced hydrogen peroxide production and 455-nm complex formation in microsomes from untreated and phenobarbital-treated rats

The following pathway for benzphetamine (Bz) metabolism in rat hepatic microsomes was established: Bz leads to norbenzphetamine (NorBz) leads to N-hydroxynorbenzphetamine leads to N-benzylethyl-alpha-phenylnitrone leads to 2-nitroso-1-phenylpropane. The last product forms a complex with cytochrome P-450 with an absorbance maximum at 455 nm. Steps 1, 2, and 4 are cytochrome P-450-dependent; Step 3 appears to involve the flavoprotein, mixed-function amine oxidase. Step 2 is partially uncoupled, producing H2O2 at approximately 3 times the rate of N-hydroxylation. Bz is oxidized to NorBz in microsomes from both untreated rats (U-microsomes) and phenobarbital (PB)-treated rats (PB-microsomes), but the 455-nm peak does not appear in U-microsomes until almost all of the Bz has been converted to NorBz; i.e., Bz inhibits the oxidation of NorBz in U- but not in PB-microsomes. The inhibition is competitive. Bz inhibits the oxidation of the nitrone to 2-nitroso-1-phenylpropane in both U- and PB-microsomes; NorBz inhibits this reaction in U-microsomes only. These results can be explained as follows. The substrate affinities of the cytochrome P-450 primarily responsible for the N-demethylation of Bz in U- and PB-microsomes differ markedly. The constitutive cytochrome(s) in U-microsomes has a high affinity for Bz; PB induces both this form and a cytochrome(s) with a lower affinity for Bz. The substrate affinities of these two cytochromes P-450 for NorBz do not differ appreciably. Thus, although both forms of cytochrome P-450 can oxidize Bz and NorBz in PB-microsomes, Bz is primarily oxidized by the constitutive form, whereas NorBz is oxidized primarily by the induced form, thereby relieving competition and increasing the over-all sequential oxidation of Bz. The nitrone appears to be oxidized exclusively by the constitutive form in both U- and PB-microsomes. The current study shows that PB induction of monooxygenase activity need not be due entirely to an increase in the amount of cytochrome P-450 or the substrate selectivity of cytochrome P-450 isozyme(s) responsible for that activity, but that, in at least one case, the metabolism of Bz, PB-induced activity can be due, at least in part, to the induction of a cytochrome P-450 isozyme that relieves substrate inhibition.