7-Benzyloxyresorufin-O-dealkylase activity as a marker for measuring cytochrome P450 CYP3A induction in mouse liver

Anal Biochem. 2010 Mar 1;398(1):104-11. doi: 10.1016/j.ab.2009.11.004. Epub 2009 Nov 10.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Anticonvulsants / pharmacology
  • Blotting, Western
  • Cytochrome P-450 CYP2B1 / metabolism*
  • Cytochrome P-450 CYP3A / metabolism*
  • Cytochrome P-450 CYP3A Inhibitors
  • Enzyme Assays / methods*
  • Male
  • Mice
  • Mice, Inbred C57BL
  • Microsomes, Liver / enzymology*
  • Oxazines / metabolism
  • Oxidation-Reduction
  • Phenytoin / pharmacology
  • RNA, Messenger / metabolism
  • Reverse Transcriptase Polymerase Chain Reaction
  • Testosterone / metabolism


  • Anticonvulsants
  • Cytochrome P-450 CYP3A Inhibitors
  • Oxazines
  • RNA, Messenger
  • Testosterone
  • Phenytoin
  • resorufin
  • Cytochrome P-450 CYP2B1
  • Cytochrome P-450 CYP3A