Effect of cytochrome P450 (CYP) inducers on caffeine metabolism in the rat Marta Kot, W³adys³awa A. Daniel Correspondence: Abstract: Our previous studies, carried out using rat cDNA-expressed cytochrome P450 (CYP) isoforms, liver microsomes and specific CYP inhibitors, showed that the 1-N- and 3-N-demethylation of caffeine at a therapeutic concentration was predominantly catalyzed by CYP1A2 and CYP2C, its 7-N-demethylation was governed by P450s of the CYP2C subfamily, while its 8-hydroxylation was spe- cifically mediated by CYP1A2. The present study was aimed at corroborating the above-described results using another experimen- tal model, i.e. a study of caffeine metabolism in the liver microsomes and specific CYP inducers. Animals received one of the following inducers: b-naphthoflavone (100 mg/kg ip for 4 days), phenobarbital (10 mg/kg for 6 days or 100 mg/kg ip for 4 days), pregnenolone 16a-carbonitrile (100 mg/kg ip for 4 days) or 15% ethanol (» 11 g/kg in drinking water for 6 days). Sixteen hours after the last dose of an inducer liver microsomes were prepared and the caffeine metabolism and CYP isoform activities (testosterone 2a-, 2b-, 6b-, 7a-, 16b-hydroxylation and warfarin 7-hydroxylation) were investigated. b-Naphthoflavone (mainly a CYP1A in- ducer and CYP2C11 inhibitor) potently accelerated the metabolism of caffeine, the effect on 7-N-demethylation being the weakest. Moreover, the influence of b-naphthoflavone on caffeine metabolism was more potent at the substrate concentration of 100 mM than 800 mM, in particular in the case of 7-N-demethylation and 8-hydroxylation. Pregnenolone-16a-carbonitrile (mainly a CYP3A in- ducer and CYP2C11 inhibitor) moderately induced 8-hydroxylation only. Phenobarbital (an inducer of CYP2B and other CYPs and a CYP2C11 inhibitor) moderately stimulated the metabolism of caffeine, but practically did not affect 7-N-demethylation. Ethanol (mainly a CYP2E1 inducer) modestly increased the rates of the N-demethylation reactions. The presently obtained data confirm the pivotal role of CYP1A2 in the metabolism of caffeine, as well as the involvement of CYP3A in the 8-hydroxylation of caffeine and that of CYP2C11 in its 7-N-demethylation. Key words: rat, liver microsomes, cytochrome P450 induction, b-naphthoflavone, phenobarbital, pregnenolone 16a-carbonitrile, ethanol, testosterone hydroxylation, warfarin 7-hydroxylation, caffeine metabolism Introduction Caffeine (1,3,7-trimethylxanthine), a purine alkaloid is oxidized in a few positions of its structure. The compound undergoes 1-N-demethylation to theobro- mine, 3-N-demethylation to paraxanthine, 7-N-deme- thylation to theophylline and 8-hydroxylation to 1,3,7-trimethyluric acid (Fig. 1). While in man 3-N- demethylation is quantitatively the main oxidation pathway, in the rat 8-hydroxylation seems to be a do- minant metabolic reaction [4, 6, 20]. 3-N-demethyl- ation to paraxanthine is regarded as a reaction specifi- cally catalyzed by CYP1A2 [2, 4, 6, 10, 24, 27]. Therefore, caffeine is often used for phenotyping of human cytochrome P450, being applied as the selecti- 296