7-Benzyloxyresorufin-O-dealkylase activity as a marker for measuring cytochrome P450 CYP3A induction in mouse liver
Section snippets
Chemicals
Testosterone, all of the resorufins (7-benzyloxy-, 7-ethoxy-, 7-methoxy-, and 7-pentoxyresorufin as well as resorufin itself), phenytoin sodium salt, and the inhibitors N-benzyl-1-aminobenzotriazole (BBT), orphenadrine, chloramphenicol, nifedipine, ketoconazole, and sulfaphenazole were obtained from Sigma–Aldrich (Deisenhofen, Germany). NADH and NADPH were obtained from Serva Electrophoresis (Heidelberg, Germany). The antibodies were obtained from the following sources: polyclonal rabbit
Influence of phenytoin on P450 expression
We initially checked the influence of the model drug phenytoin on the expression of those liver P450 subfamilies that are generally considered to be sensitive to this type of antiepileptic drug treatment, namely CYP1A, CYP2B, CYP2C, CYP3A, and CYP4A. We used antibodies known to be cross-reactive within the specified subfamily. Immunoblot investigation of liver microsomes demonstrated a strong phenytoin-dependent induction of CYP2C29 (∼13-fold), CYP3A (∼5-fold), and CYP4A (∼4-fold). CYP2B9
Discussion
In this article, we have described BROD as a valuable marker assigning CYP3A11 induction in mouse liver. This was conducted from the analysis of P450 mRNA and protein expression, correlated with the enzymatic activity data using diverse 7-alkoxyresorufins and testosterone in phenytoin-treated mice (Table 4).
The CYP3A isoforms belong to the most important P450 subfamilies in both amount and importance for drug metabolism [3]. By far, most of the drugs used in clinical practice interact with
Acknowledgments
This work was supported by grants from the Deutsche Forschungsgemeinschaft (DFG Me 1544/4 and Vo 272/7). We thank Margarethe Ditter (Department of Neuropathology, University of Freiburg) for her extraordinary technical assistance and thank Benedikt Volk for his continuous discussion, advice, and support.
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