Stable expression of guinea pig NADPH-cytochrome P450 reductase and monkey P4501A1 cDNAs in Chinese hamster cells: establishment of cell lines highly sensitive to aflatoxin B [corrected].
To the cell line (A-15) expressing monkey P4501A1, which was developed from a Chinese hamster fibroblast cell line (CHL), a guinea pig [corrected] cDNA encoding NADPH-cytochrome P450 reductase was introduced and the transformants (designated AR-10, AR-13, and AR-18) stably expressing the reductase of increased level were established. The high-level expression of the reductase was confirmed by Northern and Western blot analyses and by measurement of the reductase activity in microsomes. The transfected clones showed 4- to 11-fold higher activity of the reductase than the parental cells. These cells showed about 9-fold higher sensitivity toward aflatoxin B1 (AFB1) cytotoxicity than A-15 cells and about 330-fold more than parental CHL cells. No difference in sensitivity was observed between CHL and the CHL-derived clones (CR-50, CR-52, and CR-68) expressing high levels of the reductase alone. Increased sensitivity of AR-10 cells was also observed with respect to sterigmatocystin cytotoxicity. Furthermore, in the assay for the induction of chromosomal aberrations, these clones were highly sensitive to AFB1. These results indicate that the level of the P450 reductase existing in parental CHL cells is not sufficient for maximal activity of monkey P4501A1.
Sawada
M
M
Division of Drug Metabolism, Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan.
Kitamura
R
R
Ohgiya
S
S
Kamataki
T
T
D016604:Aflatoxin B1 / Q000494:pharmacology*; D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects*; D002869:Chromosome Aberrations; D006224:Cricetinae; D003412:Cricetulus; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D004247:DNA / Q000235:genetics / Q000378:metabolism; D006168:Guinea Pigs; D000882:Haplorhini; D008861:Microsomes / Q000201:enzymology*; D008969:Molecular Sequence Data; D009251:NADPH-Ferrihemoprotein Reductase / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids; D012333:RNA, Messenger / Q000032:analysis / Q000378:metabolism; D014162:Transfection
D012333:RNA, Messenger; D004247:DNA; D003577:Cytochrome P-450 Enzyme System; D016604:Aflatoxin B1; D009251:NADPH-Ferrihemoprotein Reductase