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.
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.
Duarte
Maria Paula
MP
Department of Genetics, Faculty of Medical Sciences, Universidade Nova de Lisboa, Rua da Junqueira 96, 1349-008 Lisboa, Portugal.
Palma
Bernardo Brito
BB
Gilep
Andrei A
AA
Laires
António
A
Oliveira
José Santos
JS
Usanov
Sergey A
SA
Rueff
José
J
Kranendonk
Michel
M
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
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