title: Stable expression of human cytochrome P450 3A4 in conjunction with human NADPH-cytochrome P450 oxidoreductase in V79 Chinese hamster cells.
abstract: 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.
authors.affiliation: Institut für Toxikologie und Umwelthygiene, Technische Universität München, Germany.
authors.forename: A
authors.initials: A
authors.lastname: Schneider
authors.forename: W A
authors.initials: WA
authors.lastname: Schmalix
authors.forename: V
authors.initials: V
authors.lastname: Siruguri
authors.forename: E M
authors.initials: EM
authors.lastname: de Groene
authors.forename: G J
authors.initials: GJ
authors.lastname: Horbach
authors.forename: B
authors.initials: B
authors.lastname: Kleingeist
authors.forename: D
authors.initials: D
authors.lastname: Lang
authors.forename: R
authors.initials: R
authors.lastname: Böcker
authors.forename: C
authors.initials: C
authors.lastname: Belloc
authors.forename: P
authors.initials: P
authors.lastname: Beaune
authors.forename: H
authors.initials: H
authors.lastname: Greim
authors.forename: J
authors.initials: J
authors.lastname: Doehmer
mesh_terms: 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
chemical_list: 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