title: Fluorescence-based screening of cytochrome P450 activities in intact cells.
abstract: Fluorimetric methods to assess cytochrome P450 (P450) activities that do not require metabolite separation have been developed. These methods make use of non- or low-fluorescent P450 substrates that produce highly fluorescent metabolites in aqueous solutions. The assays are based on the direct incubation of intact cells in culture with appropriate fluorogenic probe substrates, followed by fluorimetric quantification of the product formed and released into incubation medium. We describe a battery of fluorescence assays for rapid measurement of the activity of nine P450s involved in drug metabolism. For each individual P450 activity the probe showing the best properties (highest metabolic rates, lowest background fluorescence) has been selected. Fluorescence-based assays are highly sensitive and allow the simultaneous activity assessments of cells cultured in 96-well plates, using plate readers, with notable reductions in costs, time, and cells, thus enhancing sample throughput.
authors.affiliation: Departamento de Bioquímica y Biología Molecular, Universidad de Valencia, Valencia, Spain.
authors.forename: M Teresa
authors.initials: MT
authors.lastname: Donato
authors.forename: M José
authors.initials: MJ
authors.lastname: Gómez-Lechón
mesh_terms: D002138:Calibration; D018929:Cell Culture Techniques; D002460:Cell Line; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis / Q000378:metabolism*; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D057075:Enzyme Assays / Q000379:methods*; D004790:Enzyme Induction / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005470:Fluorometry / Q000379:methods*; D022781:Hepatocytes / Q000201:enzymology; D006801:Humans
chemical_list: D065607:Cytochrome P-450 Enzyme Inhibitors; D004791:Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System