Participation of cytochrome P450-2B and -2D isozymes in the demethylenation of methylenedioxymethamphetamine enantiomers by rats

The cytochrome P450 isozymes in rat liver microsomes that catalyze the demethylenation of methylenedioxymethamphetamine enantiomers to the corresponding dihydroxymethamphetamine were characterized. Dihydroxymethamphetamine formation in liver microsomes from male Sprague- Dawley rats exhibited multienzyme kinetics, with K(m) values in the micromolar/millimolar range. The stereoselectivity [(+)-isomer versus (-)- isomer] varied from 0.78 to 1.94 after pretreatment of the rats with phenobarbital, 3-methylcholanthrene, pregnenolone-16α-carbonitrile, or pyrazole, suggesting that different isozymes participate in the reaction. The low-K(m) demethylenation was not induced by these compounds and was not inhibited by antibodies raised against CYP2C11. Liver microsomes from female Dark-Agouti rats, a strain genetically deficient in CYP2D1, exhibited demethylenation activities that were 9% of those in microsomes from male Sprague-Dawley rats. The low-K(m) demethylenation was also inhibited by CYP2D substrates such as sparteine, bufuralol, or desipramine and was almost completely inhibited by antibodies against P450 BTL, which belongs to the CYP2D family. The high-K(m) demethylenation activity was induced by phenobarbital and pregnenolone-16α-carbonitrile and the activity in both untreated and phenobarbital-induced microsomes was suppressed by anti-CYP2B1 IgG. Experiments with IgG raised against cytochrome b₅ suggested that the hemoprotein contributed to the low-K(m) activity but not the high-K(m) activity. These results indicate that cytochrome P450 isozymes belonging to the CYP2D subfamily catalyze demethylenation with low K(m) values and that the reaction occurring with high K(m) values is likely to be mediated by members of the CYP2B family, but with the possible participation of other phenobarbital-inducible isoforms.