Mechanism of formation of 6-amino-5-(N-methylformylamino)-1-methyluracil and 3,7-dimethyluric acid from theobromine in the rat in vitro: involvement of cytochrome P-450 and a cellular thiol.

1. The involvement of glutathione (GSH) and cytochrome P-450 in the conversion of theobromine to 6-amino-5-(N-methylformylamino)-1-methyluracil (3,7-DAU) and 3,7-dimethyluric acid (3,7-DMU) has been investigated in rat liver microsomal incubations. 2. The ratio of formation of 3,7-DAU to 3,7-DMU increased with increasing GSH concentration, reaching a maximum (approximately 12:1) at 2 mM. For any given added GSH concentration the formation of 3,7-DAU plus 3,7-DMU remained constant. 3. 3,7-DAU and 3,7-DMU formation were increased approx. 12- and 1.6-fold in liver microsomes from rats treated with 3-methylcholanthrene and phenobarbitone, respectively. Cimetidine, metyrapone and SKF-525A each inhibited the conversion of theobromine to 3,7-DAU and 3,7-DMU. 4. Apparent Km and Vmax values for the combined formation of 3,7-DAU and 3,7-DMU were the same in the absence and presence of GSH, 2 mM. 5. L-Cysteine and N-acetyl-L-cysteine were as effective as GSH in causing a shift from 3,7-DMU to 3,7-DAU formation, but the non-thiol reducing agents ascorbic acid and alpha-tocopherol were ineffective. 6. Data are consistent with the hypothesis that 3,7-DAU and 3,7-DMU are derived from a common oxidized intermediate of theobromine, the formation of which is rate-limiting. The putative intermediate normally serves as a precursor to 3,7-DMU but in the presence of GSH, or some other cellular thiol, it may be reduced to give 3,7-DAU.