O-demethylation of scoparone and studies on the scoparone-induced spectral change of cytochrome P-450 in rat liver microsomes.

Scoparone (6,7-dimethoxycoumarin) is demethylated to scopoletin (7-hydroxy-6-methoxycoumarin) and isoscopoletin (6-hydroxy-7-methoxycoumarin) by the cytochrome P-450-dependent monooxygenase system of rat liver microsomes. Under the conditions used, the ratio of scopoletin to isoscopoletin was determined to 1:1.8 +/- 0.1 for microsomes from untreated rats. Based on this reaction, a direct fluorometric method for the microsomal O-demethylation activity for scoparone is described. The fluorescence of the scopoletin formed in the incubation mixture is recorded after the adjustment of the excitation and emission wavelengths to 398 and 460 nm, respectively. The fluorescence of scoparone and isoscopoletin does not interfere with the test. Pretreatment of rats with phenobarbital or polycyclic hydrocarbons (3-metylcholanthrene, benzo[a]pyrene) causes a change in the ratio of the demethylation products scopoletin to isoscopoletin which was determined to be 1:2.5 +/- 0.1 (benzo[a]pyrene or 3-methylccholanthrene) or 1:5.9 +/- 0.01 (phenobarbital) respectively, and a significant increase in the amount of microsomal O-demethylation activity. Thus the ratio of the two products varies significantly with the state of induction. The difference spectra of scoparone with liver microsomes obtained from benzo[a]pyrene- and 3-methylcholanthrene-pretreated rats show an absorption peak at 416 nm and a trough at 393 nm with an isosbestic point at 405 nm. This scoparone-induced modified Type II spectral change seems to indicate the interaction of the 6- or 7-methoxy group of scoparone with the heme ion of the 394-nm form and its conversion into a modified ferrihemochrome with a absorption peak at 416 nm. This is modified ferrihemochrome is not identical with the 418-nm form of the cytochrome P-450.