title: Development of a Salmonella tester strain sensitive to promutagenic N-nitrosamines: expression of recombinant CYP2A6 and human NADPH-cytochrome P450 reductase in S. typhimurium YG7108.
abstract: We developed a new Salmonella tester strain highly sensitive to promutagenic N-nitrosamines by introducing a plasmid carrying human cytochrome P450 2A6 (CYP2A6) and NADPH-cytochrome P450 reductase (OR) cDNA into the ada- and ogt-deficient strain YG7108. The YG7108 2A6/OR cells expressed high levels of CYP2A6 (77+/-8nmol/l) and OR (470+/-20 micromol cytochrome c reduced/min/l). The expressed CYP2A6 efficiently catalyzed coumarin 7-hydroxylation. N-Nitrosodiethylamine (NDEA), N-nitrosomethylphenylamine (NMPhA), and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) were mutagenic in the new strain in the absence of any exogenous activation system. The concentrations of promutagen that caused a two-fold increase in revertants were 7.1, 0.14, and 1.4 microM for NDEA, NMPhA, and NNK, respectively. YG7108 2A6/OR cells showed about 10- and 100-fold higher sensitivity to NDEA and NNK, respectively, than parental YG7108 cells assayed in the presence of rat liver S9 (final concentration, 21% (v/v)). Parental YG7108 cells did not detect NMPhA mutagenicity even in the presence of rat liver S9. We believe that this is the first demonstration that CYP2A6 is responsible for the metabolic activation of NMPhA. The established tester strain may be useful to predict human activation of N-nitrosamine promutagens.
authors.affiliation: Laboratory of Drug Metabolism, Division of Pharmacobio-dynamics, Graduate School of Pharmaceutical Sciences, Hokkaido University, Kita-ku N12W6, Sapporo 060-0812, Japan.
authors.forename: H
authors.initials: H
authors.lastname: Kushida
authors.forename: K
authors.initials: K
authors.lastname: Fujita
authors.forename: A
authors.initials: A
authors.lastname: Suzuki
authors.forename: M
authors.initials: M
authors.lastname: Yamada
authors.forename: T
authors.initials: T
authors.lastname: Nohmi
authors.forename: T
authors.initials: T
authors.lastname: Kamataki
mesh_terms: D001189:Aryl Hydrocarbon Hydroxylases; D001711:Biotransformation; D065637:Cytochrome P-450 CYP2A6; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism; D004052:Diethylnitrosamine / Q000633:toxicity*; D004305:Dose-Response Relationship, Drug; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology; D006899:Mixed Function Oxygenases / Q000235:genetics* / Q000378:metabolism; D009152:Mutagenicity Tests / Q000379:methods*; D009153:Mutagens / Q000633:toxicity*; D009251:NADPH-Ferrihemoprotein Reductase / Q000235:genetics*; D009602:Nitrosamines / Q000633:toxicity*; D012333:RNA, Messenger / Q000032:analysis; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012486:Salmonella typhimurium / Q000145:classification / Q000201:enzymology / Q000235:genetics*; D012680:Sensitivity and Specificity; D013045:Species Specificity
chemical_list: D009153:Mutagens; D009602:Nitrosamines; D012333:RNA, Messenger; D011994:Recombinant Proteins; D004052:Diethylnitrosamine; C016583:4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone; D003577:Cytochrome P-450 Enzyme System; C014102:N-methyl-N-nitrosoaniline; D006899:Mixed Function Oxygenases; D001189:Aryl Hydrocarbon Hydroxylases; C585710:CYP2A6 protein, human; D065637:Cytochrome P-450 CYP2A6; D009251:NADPH-Ferrihemoprotein Reductase