The stimulatory role of human cytochrome b5 in the bioactivation activities of human CYP1A2, 2A6 and 2E1: A new cell expression system to study cytochrome P450 mediated biotransformation

Maria Paula Duarte, Bernardo Brito Palma, Andrei A. Gilep, António Laires, José Santos Oliveira, Sergey A. Usanov, José Rueff, Michel Kranendonk

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20 Citations (Scopus)

Abstract

Cytochrome b5 (b5) is increasingly recognized to be of importance for specific cytochrome P450 (CYP) activities. We developed human b5/CYP-competent mutagenicity tester bacteria to study the role of b5 in the bioactivation activity of human CYP. These new tester bacteria were derived from the previously engineered human CYP-competent Escherichia coli K12 tester strain MTC, containing a bi-plasmid system for the co-expression of a specific CYP form (CYP1A2, 2A6 or 2E1) with human b 5, and human NADPH cytochrome P450 reductase (RED), resulting in the strain BTC-b5-1A2, BTC-b5-2A6 and BTC-b5-2E1, respectively. The relative content of b5 with CYP and RED in these three BTC-b5-CYP strains demonstrated physiologically relevant co-expression levels and typical CYP-specific activities could be determined with their specific chemical probes. These strains were applied in mutagenicity assays along with their corresponding b5-void strains to determine the effect of b5 on the CYP1A2-, CYP2A6- and CYP2E1-mediated bioactivation of several promutagens. For CYP1A2, of the 5 compounds tested [2-aminoanthracene (2AA), 1-aminopyrene, 6-aminochrysene, 2-amino-3- methylimidazo(4,5-f)quinoline and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)], only the mutagenicity of 2AA was slightly increased (∼1.5-fold) in the presence of b5. The CYP2E1- and CYP2A6-dependent mutagenicity of N-nitrosodiethylamine increased ∼3- and 23-fold, respectively when the bacteria contained b5. The CYP2A6-mediated mutagenicity of NNK increased ∼9-fold when co-expressed with b5. The stimulatory effect of b5 on the bioactivation of N-nitrosodi-n-propylamine was most striking. The mutagenicity of this procarcinogen was completely dependent on the co-expression of b5 with CYP2A6 or CYP2E1. This demonstrates the prominent role of b5 in the bioactivation of this carcinogen.

Original languageEnglish
Pages (from-to)93-100
Number of pages8
JournalMutagenesis
Volume20
Issue number2
DOIs
Publication statusPublished - 1 Mar 2005

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