BIOTRANSFORMATION OF METHYLXANTHINES IN MAMMALIAN-CELL LINES GENETICALLY-ENGINEERED FOR EXPRESSION OF SINGLE CYTOCHROME-P450 ISOFORMS - ALLOCATION OF METABOLIC PATHWAYS TO ISOFORMS AND INHIBITORY EFFECTS OF QUINOLONES

V79 Chinese hamster cells genetically engineered for stable expression of single forms of rat cytochromes P450IA1, P450IA2, P450IIB1, human P450IA2, and rat liver epithelial cells expressing murine P450IA2 were used to allocate metabolic pathways of methylxanthines to specific is oforms and to test the suitability of such cell lines for investigatio ns on drug interactions occurring at the cytochrome expressed. The cel l lines were exposed to caffeine and/or theophylline and concentration s of metabolites formed in the medium were determined by HPLC. Caffein e was metabolized by human, rat and murine P450IA2, resulting in the f ormation of four primary demethylated and hydroxylated metabolites. Ho wever, there were differences in the relative amounts of the metabolit es. The human and the mouse P450IA2 isoforms predominantly mediated 3- demethylation of caffeine. The rat cytochrome P450IA2 mediated both 3- demethylation and 1-demethylation of caffeine to a similar extent. The results support the hypothesis that caffeine plasma clearance is a sp ecific in vivo probe for determining human P450IA2 activity. Addition of the quinolone antibiotic agents pipemidic acid or pefloxacin, both known to inhibit caffeine metabolism in vivo and in human liver micros omes, reduced formation rates of all metabolites of caffeine in cells expressing rat and human P450IA2. Theophylline was mainly metabolized via 8-hydroxylation. All cell lines tested were able to carry out this reaction, with highest activities in cell lines expressing rat or hum an P450IA2, or rat P450IA1.