Cytochrome P450 isoform selectivity in human hepatic theobromine metabolism.
OBJECTIVE
The plasma clearance of theobromine (TB; 3,7-dimethylxanthine) is known to be induced in cigarette smokers. To determine whether TB may serve as a model substrate for cytochrome P450 (CYP) 1A2, or possibly other isoforms, studies were undertaken to identify the individual human liver microsomal CYP isoforms responsible for the conversion of TB to its primary metabolites.


METHODS
The kinetics of formation of the primary TB metabolites 3-methylxanthine (3-MX), 7-methylxanthine (7-MX) and 3,7-dimethyluric acid (3,7-DMU) by human liver microsomes were characterized using a specific hplc procedure. Effects of CYP isoform-selective xenobiotic inhibitor/substrate probes on each pathway were determined and confirmatory studies with recombinant enzymes were performed to define the contribution of individual isoforms to 3-MX, 7-MX and 3,7-DMU formation.


RESULTS
The CYP1A2 inhibitor furafylline variably inhibited (0-65%) 7-MX formation, but had no effect on other pathways. Diethyldithiocarbamate and 4-nitrophenol, probes for CYP2E1, inhibited the formation of 3-MX, 7-MX and 3,7-DMU by approximately 55-60%, 35-55% and 85%, respectively. Consistent with the microsomal studies, recombinant CYP1A2 and CYP2E1 exhibited similar apparent Km values for 7-MX formation and CYP2E1 was further shown to have the capacity to convert TB to both 3-MX and 3,7-DMU.


CONCLUSIONS
Given the contribution of multiple isoforms to 3-MX and 7-MX formation and the negligible formation of 3,7-DMU in vivo, TB is of little value as a CYP isoform-selective substrate in humans.
Gates
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Department of Clinical Pharmacology, Flinders Medical Centre and Flinders University School of Medicine, Bedford Park, SA, Australia.
Miners
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D000818:Animals; D019556:COS Cells; D019388:Cytochrome P-450 CYP1A2 / Q000235:genetics / Q000378:metabolism*; D065609:Cytochrome P-450 CYP1A2 Inhibitors; D019392:Cytochrome P-450 CYP2E1 / Q000235:genetics / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015870:Gene Expression; D006801:Humans; D007700:Kinetics; D008862:Microsomes, Liver / Q000187:drug effects / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D013805:Theobromine / Q000378:metabolism*; D014527:Uric Acid / Q000031:analogs & derivatives / Q000378:metabolism; D014970:Xanthines / Q000378:metabolism
D065609:Cytochrome P-450 CYP1A2 Inhibitors; D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D011993:Recombinant Fusion Proteins; D014970:Xanthines; D014527:Uric Acid; C064273:7-methylxanthine; D019392:Cytochrome P-450 CYP2E1; D019388:Cytochrome P-450 CYP1A2; C066065:3,7-dimethyluric acid; D013805:Theobromine; C029703:3-methylxanthine