Stable expression and coexpression of human cytochrome P450 oxidoreductase and cytochrome P450 1A2 in V79 Chinese hamster cells: sensitivity to quinones and biotransformation of 7-alkoxyresorufins and triazines.
V79 Chinese hamster cell lines were genetically engineered for the stable expression of human NADPH-cytochrome P450 oxidoreductase (CYPOR) alone or for the combined expression of CYPOR and human cytochrome P450 1A2 (CYP1A2). As determined by immunoblotting, the expression level of CYP1A2 in the latter cell line was found to be the same as in a previously constructed V79 cell line expressing CYP1A2 only. The heterologous expression of CYPOR in V79 cells resulted in increased sensitivity to quinone-type cytotoxins, e.g. duroquinone and menadione, that exert their toxicity primarily through the production of reactive oxygen species during redox cycling. The metabolic properties of the cell line expressing both CYPOR and CYP1A2 were characterized regarding dealkylation and deethylation of 7-alkoxyresorufins and sulfoxidation of the triazine derivatives ametryne and terbutryne, in comparison with the cell line expressing only CYP1A2. Increased CYPOR activity impaired the CYP1A2-dependent fluorometric resorufin assay, presumably by conversion of the 7-alkoxyresorufins and resorufin to their one-electron-reduced semiquinoneimine forms. The CYP1A2-dependent metabolism of the triazine derivatives ametryne and terbutryne was moderately enhanced by increased CYPOR activity. Interestingly, with CYPOR overexpression sulfoxidation was increased 2-3-fold, compared with N-deethylation, with a 1.3-1.9-fold increase. Thus, the level of CYPOR not only had an influence on CYP1A2 activity rates but also affected the relative proportions of metabolites in CYP1A2-specific metabolite profiles.
Schmalix
W A
WA
Technische Universität München, Institute für Toxikologie und Umwelthygiene, Germany.
Lang
D
D
Schneider
A
A
Bocker
R
R
Greim
H
H
Doehmer
J
J
D000818:Animals; D001711:Biotransformation; D016466:CHO Cells; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D006224:Cricetinae; D019388:Cytochrome P-450 CYP1A2 / Q000096:biosynthesis*; D005822:Genetic Vectors; D006801:Humans; D015151:Immunoblotting; D009247:NADH, NADPH Oxidoreductases / Q000096:biosynthesis*; D009251:NADPH-Ferrihemoprotein Reductase / Q000096:biosynthesis; D010078:Oxazines / Q000378:metabolism*; D011809:Quinones / Q000494:pharmacology*; D012333:RNA, Messenger / Q000096:biosynthesis; D014162:Transfection; D014227:Triazines / Q000494:pharmacology*
D010078:Oxazines; D011809:Quinones; D012333:RNA, Messenger; D014227:Triazines; C044943:pentoxyresorufin; D019388:Cytochrome P-450 CYP1A2; D009247:NADH, NADPH Oxidoreductases; D009251:NADPH-Ferrihemoprotein Reductase