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GENTEST Corporation (E.L.C., C.L.C., B.W.P.);
Laboratory of
Molecular Carcinogenesis (F.J.G.), National Cancer Institute, National
Institutes of Health;
Faculty of Pharmaceutical Sciences (T.K.H.C.),
University of British Columbia; and the
Division of Cell and Molecular
Biology (D.J.W.), Department of Biology, Boston University
The level of expression and interindividual variation in human
hepatic microsomal cytochrome P450 (CYP) 2B6 was characterized using a
polyclonal antibody (WB-2B6) raised against rat CYP2B1. Immunoblot
analysis using cDNA-expressed human CYPs revealed strong cross-reactivity of this antibody with CYP2B6 (limit of detection < 0.05 pmol) and only minor cross-reactivities with human CYP2A6, CYP2D6, and CYP2E1, all of which could be resolved from CYP2B6 by
sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Analysis of
human liver microsomes using this antibody revealed immunodetectable
CYP2B6 protein in a majority of individual liver samples, with levels
up to 74 pmol/mg protein in the CYP2B6-positive samples. Kinetic
analysis of cDNA-expressed CYPs identified many of these enzymes as
catalysts of 7-ethoxy-4-trifluoromethylcoumarin (7EFC)
O-deethylation, but with significantly different apparent KM values (CYP1A2 < CYP2B6 ~ CYP1A1 < CYP2C19 < CYP2C9 < CYP2E1 < CYP2A6).
By assaying liver microsomal 7EFC O-deethylase activity at
a low 7EFC concentration (5 µM) and preincubating human liver microsomes with anti-CYP1A, anti-CYP2C, and anti-CYP2E1 antibodies, we
were able to monitor CYP2B6-dependent 7EFC O-deethylase
activity in a panel of 17 human liver microsomes and observe a
significant correlation (r2 = 0.80) between
this activity and CYP2B6 protein content. The ability of CYP2B6 to
activate prodrugs and procarcinogens was examined using gene locus
mutation assays in CYP2B6-expressing human lymphoblast cells.
CYP2B6-expressing cells were found to be more sensitive than control
cells to the cytotoxicity and mutagenicity of cyclophosphamide,
aflatoxin B1, and
4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone. CYP2B6 is thus a widely
expressed human liver microsomal CYP that can contribute to a broad
range of drug metabolism and procarcinogen activation reactions.
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