Co-expression of human cytochrome P4501A1 (CYP1A1) variants and human NADPH-cytochrome P450 reductase in the baculovirus/ insect cell system |
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Authors: | D Schwarz P Kisselev H Honeck I Cascorbi W-H Schunck I Roots |
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Institution: | Institute of Clinical Pharmacology, University Medical Centre Charite, Humboldt University of Berlin, 10098 Berlin, Germany |
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Abstract: | 1. Three human cytochrome P4501A1 (CYP1A1) variants, wild-type (CYP1A1.1), CYP1A1.2 (I462V) and CYP1A1.4 (T461N), were co-expressed with human NADPHP450 reductase (OR) in Spodoptera frugiperda (Sf9) insect cells by baculovirus coinfection to elaborate a suitable system for studying the role of CYP1A1 polymorphism in the metabolism of exogenous and endogenous substrates. 2. A wide range of conditions was examined to optimize co-expression with regard to such parameters as relative multiplicity of infection (MOI), time of harvest, haem precursor supplementation and post-translational stabilization. Under optimized conditions, almost identical expression levels and molar OR/CYP1A1 ratios (20:1) were attained for all CYP1A1 variants. 3. Microsomes isolated from co-infected cells demonstrated ethoxyresorufin deethylase activities (nmol/min-1 nmol-1 CYP1A1) of 16.0 (CYP1A1.1), 20.5 (CYP1A1.2) and 22.5 (CYP1A1.4). Pentoxyresorufin was dealkylated ~ 10-20 times slower with all enzyme variants. 4. All three CYP1A1 variants were active in metabolizing the precarcinogen benzoa]pyrene (Ba]P), with wild-type enzyme showing the highest activity, followed by CYP1A1.4 (60%) and CYP1A1.2 (40%). Each variant produced all major metabolites including B\a]P-7,8-dihydrodiol, the precursor of the ultimate carcinogenic species. 5. These studies demonstrate that the baculovirus-mediated co-expression-by-coinfection approach all CYP1A1 variants yields functionally active enzyme systems with similar molar OR/CYP1A1 ratios, thus providing suitable preconditions to examine the metabolism of and environmental chemicals by the different CYP1A1 variants. |
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