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 sta ble expression of human NADPH-cytochrome P450 oxidoreductase (CYPOR) a lone or for the combined expression of CYPOR and human cytochrome P450 1A2 (CYP1A2). As determined by immunoblotting, the expression level o f CYP1A2 in the latter cell line was found to be the same as in a prev iously constructed V79 cell line expressing CYP1A2 only. The heterolog ous expression of CYPOR in V79 cells resulted in increased sensitivity to quinone-type cytotoxins, e.g. duroquinone and menadione, that exer t their toxicity primarily through the production of reactive oxygen s pecies during redox cycling. The metabolic properties of the cell line expressing both CYPOR and CYP1A2 were characterized regarding dealkyl ation and deethylation of 7-alkoxyresorufins and sulfoxidation of the triazine derivatives ametryne and terbutryne, in comparison with the c ell line expressing only CYP1A2. Increased CYPOR activity impaired the CYP1A2-dependent fluorometric resorufin assay, presumably by conversi on of the 7-alkoxyresorufins and resorufin to their one-electron-reduc ed semiquinoneimine forms. The CYP1A2-dependent metabolism of the tria zine derivatives ametryne and terbutryne was moderately enhanced by in creased CYPOR activity, interestingly, with CYPOR overexpression sulfo xidation 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 influe nce on CYP1A2 activity rates but also affected the relative proportion s of metabolites in CYP1A2-specific metabolite profiles.