title: 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.
abstract: Cytochrome b(5) (b(5)) is increasingly recognized to be of importance for specific cytochrome P450 (CYP) activities. We developed human b(5)/CYP-competent mutagenicity tester bacteria to study the role of b(5) 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-b(5)-1A2, BTC-b(5)-2A6 and BTC-b(5)-2E1, respectively. The relative content of b(5) with CYP and RED in these three BTC-b(5)-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 b(5)-void strains to determine the effect of b(5) 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 ( approximately 1.5-fold) in the presence of b(5). The CYP2E1- and CYP2A6-dependent mutagenicity of N-nitrosodiethylamine increased approximately 3- and 23-fold, respectively when the bacteria contained b(5). The CYP2A6-mediated mutagenicity of NNK increased approximately 9-fold when co-expressed with b(5). The stimulatory effect of b(5) on the bioactivation of N-nitrosodi-n-propylamine was most striking. The mutagenicity of this procarcinogen was completely dependent on the co-expression of b(5) with CYP2A6 or CYP2E1. This demonstrates the prominent role of b(5) in the bioactivation of this carcinogen.
authors.affiliation: Department of Genetics, Faculty of Medical Sciences, Universidade Nova de Lisboa, Rua da Junqueira 96, 1349-008 Lisboa, Portugal.
authors.forename: Maria Paula
authors.initials: MP
authors.lastname: Duarte
authors.forename: Bernardo Brito
authors.initials: BB
authors.lastname: Palma
authors.forename: Andrei A
authors.initials: AA
authors.lastname: Gilep
authors.forename: António
authors.initials: A
authors.lastname: Laires
authors.forename: José Santos
authors.initials: JS
authors.lastname: Oliveira
authors.forename: Sergey A
authors.initials: SA
authors.lastname: Usanov
authors.forename: José
authors.initials: J
authors.lastname: Rueff
authors.forename: Michel
authors.initials: M
authors.lastname: Kranendonk
mesh_terms: D001189:Aryl Hydrocarbon Hydroxylases / Q000235:genetics / Q000378:metabolism*; D001711:Biotransformation; D002273:Carcinogens / Q000378:metabolism; D019388:Cytochrome P-450 CYP1A2 / Q000235:genetics / Q000378:metabolism*; D065637:Cytochrome P-450 CYP2A6; D019392:Cytochrome P-450 CYP2E1 / Q000235:genetics / Q000378:metabolism*; D015786:Cytochromes b5 / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D048168:Escherichia coli K12 / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000378:metabolism*; D009152:Mutagenicity Tests; D009153:Mutagens / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015262:Xenobiotics / Q000378:metabolism
chemical_list: D002273:Carcinogens; D009153:Mutagens; D011994:Recombinant Proteins; D015262:Xenobiotics; D015786:Cytochromes b5; D006899:Mixed Function Oxygenases; D019392:Cytochrome P-450 CYP2E1; D001189:Aryl Hydrocarbon Hydroxylases; C585710:CYP2A6 protein, human; D019388:Cytochrome P-450 CYP1A2; D065637:Cytochrome P-450 CYP2A6; D009251:NADPH-Ferrihemoprotein Reductase