Experimental approaches to evaluate activities of cytochromes P450 3A

Lucie Bořek-Dohalská,Jiří Hudeček,Petr Hodek,Marie Stiborová

doi:10.2478/v10102-010-0032-0

Abstract

Cytochrome P450 (CYP) is a heme protein oxidizing various xenobiotics, as well as endogenous substrates. Understanding which CYP enzymes are involved in metabolic activation and/or detoxication of different compounds is important in the assessment of an individual's susceptibility to the toxic action of these substances. Therefore, investigation which of several in vitro experimental models are appropriate to mimic metabolism of xenobiotics in organisms is the major challenge for research of many laboratories. The aim of this study was to evaluate the efficiency of different in vitro systems containing individual enzymes of the mixed-function monooxygenase system to oxidize two model substrates of CYP3A enzymes, exogenous and endogenous compounds, α-naphtoflavone (α-NF) and testosterone, respectively. Several different enzymatic systems containing CYP3A enzymes were utilized in the study: (i) human hepatic microsomes rich in CYP3A4, (ii) hepatic microsomes of rabbits treated with a CYP3A6 inducer, rifampicine, (iii) microsomes of Baculovirus transfected insect cells containing recombinant human CYP3A4 and NADPH:CYP reductase with or without cytochrome b5 (Supersomes™), (iv) membranes isolated from of Escherichia coli, containing recombinant human CYP3A4 and cytochrome b5, and (v) purified human CYP3A4 or rabbit CYP3A6 reconstituted with NADPH:CYP reductase with or without cytochrome b5 in liposomes. The most efficient systems oxidizing both compounds were Supersomes™ containing human CYP3A4 and cytochrome b5. The results presented in this study demonstrate the suitability of the supersomal CYP3A4 systems for studies investigating oxidation of testosterone and α-NF in vitro.

Highlights

The cytochrome P450s (CYP) are a family of hemoprotein enzymes that play important roles in the metabolism of drugs and carcinogens, as well as endogenous compounds such as prostaglandins, fatty acids and steroids (Gonzalez and Gelboin, 1992, Ortiz de Montellano 1995).CYP3A is one of the major subfamilies expressed in human livers and is found at high levels in the intestinal tract (Hosea et al, 2000)
Several different enzymatic systems containing CYP3A enzymes were utilized in the study: (i) human hepatic microsomes rich in CYP3A4, (ii) hepatic microsomes of rabbits treated with a CYP3A6 inducer, rifampicine, (iii) microsomes of Baculovirus transfected insect cells containing recombinant human CYP3A4 and NADPH:Cytochrome P450 (CYP) reductase with or without cytochrome b5 (SupersomesTM), (iv) membranes isolated from of Escherichia coli, containing recombinant human CYP3A4 and cytochrome b5, and (v) purified human CYP3A4 or rabbit CYP3A6 reconstituted with NADPH:CYP reductase with or without cytochrome b5 in liposomes
Several different systems containing CYP3A enzymes were utilized to investigate oxidation of testosterone and α-NF in vitro: (i) human hepatic microsomes rich in CYP3A4, (ii) hepatic microsomes of rabbit treated with CYP3A6 inducer, rifampicine, (iii) microsomes of Baculovirus transfected insect cells containing recombinant human CYP3A4 and NADPH:CYP reductase with or without cytochrome b5 (SupersomesTM), (iv) membranes isolated from of Escherichia coli, containing human CYP3A4 with or without cytochrome b5, and (v) purified human CYP3A4 or rabbit CYP3A6 reconstituted with NADPH:CYP reductase with or without cytochrome b5 in liposomes

Summary

Introduction

CYP3A is one of the major subfamilies expressed in human livers and is found at high levels in the intestinal tract (Hosea et al, 2000) This enzyme oxidizes endogenous and exogeneous compounds as well as over half of the drugs in therapeutic use (Hosea et al, 2000). During the past decade understanding of the mechanism of CYP3A4 cooperativity has progressed from a static model with multiple binding sites (Domanski et al, 1998, Shou et al, 1994, Tsalkova et al, 2007, Ueng et al, 1997) to more complex dynamic model suggesting effector-induced conformational rearrangements of the enzyme along with multiple ligand binding (Atkins et al, 2001, Isin and Guengerich 2006)

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