Stable expression of human cytochrome P450 3A4 in conjunction with human NADPH-cytochrome P450 oxidoreductase in V79 Chinese hamster cells.
V79 Chinese hamster cells were constructed for stable expression of human cytochrome P450 3A4 with and without coexpression of human NADPH-cytochrome P450 oxidoreductase. Expression of the cDNAs was shown by Northern and Western analyses. Activity was tested by 6 beta-hydroxylation of testosterone for cytochrome P450 3A4 and by cytochrome c reduction for NADPH-cytochrome P450 reductase. Five V79 cell lines were obtained expressing cytochrome P450 3A4, human NADPH-cytochrome P450 oxidoreductase, and both. Cytochrome P450 3A4 activity depended highly on cytochrome P450 reductase activity, with lowest activity when only the parental Chinese hamster cytochrome P450 reductase was present, 5- and 10-fold higher when coexpressed with the human NADPH-cytochrome P450 reductase. Correspondingly, cytotoxic and genotoxic potency of aflatoxin B1 was increased by orders of magnitudes when human cytochrome P450 3A4 was coexpressed with the human NADPH-cytochrome P450 reductase. The effect of NADPH-cytochrome P450 reductase coexpression on cytochrome P450 3A4 activity was also tested by nifedipine oxidation and midazolam hydroxylation. Nifedipine oxidation was increased about 10-fold, 1-hydroxylation of midazolam and 4-hydroxylation of midazolam were increased 15-fold.
Schneider
A
A
Institut für Toxikologie und Umwelthygiene, Technische Universität München, Germany.
Schmalix
W A
WA
Siruguri
V
V
de Groene
E M
EM
Horbach
G J
GJ
Kleingeist
B
B
Lang
D
D
Böcker
R
R
Belloc
C
C
Beaune
P
P
Greim
H
H
Doehmer
J
J
D016604:Aflatoxin B1 / Q000633:toxicity; D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism*; D018076:DNA, Complementary / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D015162:Micronucleus Tests; D006899:Mixed Function Oxygenases / Q000235:genetics* / Q000378:metabolism*; D009251:NADPH-Ferrihemoprotein Reductase / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D018076:DNA, Complementary; D011994:Recombinant Proteins; D003577:Cytochrome P-450 Enzyme System; D016604:Aflatoxin B1; D006899:Mixed Function Oxygenases; C104464:CYP3A protein, human; D051544:Cytochrome P-450 CYP3A; D009251:NADPH-Ferrihemoprotein Reductase