title: 7-Benzyloxyresorufin-O-dealkylase activity as a marker for measuring cytochrome P450 CYP3A induction in mouse liver.
abstract: The cytochrome P450 subfamily CYP3A belongs to the most important detoxification enzymes. Because the main CYP3A isoforms are not polymorphic and therefore detract themselves from genetic screening as a potent prediction marker for drug metabolism or induction effects, effective in vitro testing of a putative drug-CYP3A interaction is indicated. We used mouse liver microsomes treated with the model drug phenytoin to set up an effective and reliable in vitro test system. A metabolic assay analyzing 7-alkoxyresorufin-O-dealkylation showed specific CYP3A-dependent 7-benzyloxyresorufin oxidation (BROD). This was confirmed by testing other alkoxyresorufins (7-ethoxy-, 7-methoxy-, and 7-pentoxyresorufin) in mice and correlation of the data with testosterone 6beta-hydroxylation and a plethora of isoform-specific chemical inhibitors (orphenadrine, chloramphenicol, nifedipine, ketoconazole, and sulfaphenazole). Isoform-specific expression and induction of CYP3A11 in mouse liver was tested by RNase protection assay, reverse transcription polymerase chain reaction (RT-PCR), and immunoblot. With the BROD assay, we could clearly dissect CYP3A11 from other P450s induced by phenytoin-like CYP2C29, CYP2B9, CYP1A1, and CYP4A. We conclude that the BROD assay is a specific tool to assign CYP3A induction by drugs or other chemicals, at least in a mouse model system.
authors.affiliation: Baker IDI Heart and Diabetes Institute, Melbourne, Vic. 8008, Australia. sohn4090@kribb.re.kr
authors.forename: Christoph E
authors.initials: CE
authors.lastname: Hagemeyer
authors.forename: Carolin
authors.initials: C
authors.lastname: Bürck
authors.forename: Ricarda
authors.initials: R
authors.lastname: Schwab
authors.forename: Rolf
authors.initials: R
authors.lastname: Knoth
authors.forename: Ralf P
authors.initials: RP
authors.lastname: Meyer
mesh_terms: D000818:Animals; D000927:Anticonvulsants / Q000494:pharmacology; D015153:Blotting, Western; D019362:Cytochrome P-450 CYP2B1 / Q000378:metabolism*; D051544:Cytochrome P-450 CYP3A / Q000378:metabolism*; D065692:Cytochrome P-450 CYP3A Inhibitors; D057075:Enzyme Assays / Q000379:methods*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008862:Microsomes, Liver / Q000201:enzymology*; D010078:Oxazines / Q000378:metabolism; D010084:Oxidation-Reduction; D010672:Phenytoin / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013739:Testosterone / Q000378:metabolism
chemical_list: D000927:Anticonvulsants; D065692:Cytochrome P-450 CYP3A Inhibitors; D010078:Oxazines; D012333:RNA, Messenger; D013739:Testosterone; D010672:Phenytoin; C014180:resorufin; D019362:Cytochrome P-450 CYP2B1; D051544:Cytochrome P-450 CYP3A