A new technique for assaying cytochrome P450 enzyme activity in a single cell.
A new microspectrofluorometric technique for measuring the ethoxyresorufin-O-deethylase (EROD) activity of cytochrome P450 (CYP)1A1 in single living cells is described. The system, which uses a perfusion chamber and an HPLC pump, allowed cells to be stained, fixed, blocked, and washed by injecting each treatment solution into the on-line carrier stream of buffer from the sampling block of the HPLC pump. After addition of the substrate 7-ethoxyresorufin, the fluorescence intensity of the metabolite resorufin was measured in individual cells. Fluorescence intensity steeply increased to a unique peak for each cell and then decreased to the basal level. Furthermore, CYP1A1 in each cell was stained with its antibody and quantified using the fluorescence intensity of an FITC-conjugated secondary antibody. EROD activity was normalized using the FITC fluorescence. The results show that the initial slopes and peak values of resorufin production by the cells were dependent on the CYP1A1 level. Treatment of hepatocytes with two nonspecific P450 inhibitors, cimetidine and SKF-525A, suppressed EROD activity.
Taira
Z
Z
Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Tokushima 770-8514, Japan. tairaz@ph.bunri-u.ac.jp
Yamase
D
D
Ueda
Y
Y
D000818:Animals; D002927:Cimetidine / Q000494:pharmacology; D019363:Cytochrome P-450 CYP1A1 / Q000032:analysis / Q000037:antagonists & inhibitors / Q000276:immunology / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000032:analysis* / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D022781:Hepatocytes / Q000166:cytology / Q000201:enzymology*; D066298:In Vitro Techniques; D008297:Male; D008856:Microscopy, Fluorescence; D010078:Oxazines / Q000378:metabolism; D011335:Proadifen / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D013050:Spectrometry, Fluorescence / Q000379:methods
D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D010078:Oxazines; C007649:ethoxyresorufin; D002927:Cimetidine; D003577:Cytochrome P-450 Enzyme System; D011335:Proadifen; D019363:Cytochrome P-450 CYP1A1; D016650:Fluorescein-5-isothiocyanate