Elsevier

Analytical Biochemistry

Volume 398, Issue 1, 1 March 2010, Pages 104-111
Analytical Biochemistry

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

https://doi.org/10.1016/j.ab.2009.11.004Get rights and content

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 6β-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.

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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