Construction of Salmonella typhimurium YG7108 strains, each coexpressing a form of human cytochrome P450 with NADPH-cytochrome P450 reductase.
A series of Salmonella typhimurium (S. typhimurium) YG7108 strains, each coexpressing a form of human cytochrome P450 (CYP) (CYP1A1, CYP1A2, CYP1B1, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP3A4, or CYP3A5) together with human NADPH-cytochrome P450 reductase (OR), was established. The parental S. typhimurium YG7108, derived from TA1535, lacks two O(6)-methylguanine-DNA methyltransferase genes, ada and ogt, and is highly sensitive to the mutagenicity of alkylating agents. The expression levels of CYP holo-protein in the genetically engineered S. typhimurium YG7108 cells, determined by carbon monoxide (CO) difference spectra, ranged from 62 nmol/L culture for CYP2C19 to 169 nmol/L culture for CYP3A4. The expression level of the OR varied, depending on the form of CYP coexpressed, and ranged from 214 to 1029 units/L culture. Each form of CYP expressed in the S. typhimurium YG7108 cells catalyzed the oxidation of a representative substrate at an efficient rate. The rates appeared comparable to the reported activities of CYP expressed in human liver microsomes or CYP in other heterologous systems, indicating that the OR was sufficiently expressed to support the catalytic activity of CYP. These S. typhimurium strains may be useful not only for predicting the metabolic activation of promutagens catalyzed by human CYP but also for identifying the CYP form involved.
Fujita
K
K
Laboratory of Drug Metabolism, Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan.
Nakayama
K
K
Yamazaki
Y
Y
Tsuruma
K
K
Yamada
M
M
Nohmi
T
T
Kamataki
T
T
D000477:Alkylating Agents / Q000633:toxicity; D001189:Aryl Hydrocarbon Hydroxylases; D003374:Coumarins / Q000378:metabolism; D019363:Cytochrome P-450 CYP1A1 / Q000235:genetics; D019388:Cytochrome P-450 CYP1A2 / Q000235:genetics; D065633:Cytochrome P-450 CYP1B1; D065731:Cytochrome P-450 CYP2C19; D065727:Cytochrome P-450 CYP2C8; D065729:Cytochrome P-450 CYP2C9; D019389:Cytochrome P-450 CYP2D6; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism; D004008:Diclofenac / Q000633:toxicity; D004983:Ethanolamines / Q000378:metabolism; D005818:Genetic Engineering / Q000379:methods; D006801:Humans; D008874:Midazolam / Q000378:metabolism / Q000633:toxicity; D006899:Mixed Function Oxygenases / Q000235:genetics; D009152:Mutagenicity Tests / Q000379:methods; D009249:NADP / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase / Q000235:genetics* / Q000378:metabolism; D009602:Nitrosamines / Q000633:toxicity; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012486:Salmonella typhimurium / Q000235:genetics*; D039181:Steroid 16-alpha-Hydroxylase; D013250:Steroid Hydroxylases / Q000235:genetics
D000477:Alkylating Agents; D003374:Coumarins; D004983:Ethanolamines; D009602:Nitrosamines; D011994:Recombinant Proteins; D004008:Diclofenac; D009249:NADP; C010831:bufuralol; D003577:Cytochrome P-450 Enzyme System; C030123:coumarin; D006899:Mixed Function Oxygenases; D013250:Steroid Hydroxylases; C450260:CYP2C9 protein, human; D065729:Cytochrome P-450 CYP2C9; D001189:Aryl Hydrocarbon Hydroxylases; C504573:CYP1A2 protein, human; C585496:CYP1B1 protein, human; C045793:CYP2C19 protein, human; C450259:CYP2C8 protein, human; C104464:CYP3A protein, human; C510164:CYP3A5 protein, human; D019363:Cytochrome P-450 CYP1A1; D019388:Cytochrome P-450 CYP1A2; D065633:Cytochrome P-450 CYP1B1; D065731:Cytochrome P-450 CYP2C19; D065727:Cytochrome P-450 CYP2C8; D019389:Cytochrome P-450 CYP2D6; D051544:Cytochrome P-450 CYP3A; D039181:Steroid 16-alpha-Hydroxylase; C510163:CYP3A4 protein, human; D009251:NADPH-Ferrihemoprotein Reductase; D008874:Midazolam