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.
KeywordsCYP1A1 metabolism microspectrofluorometry single cell
high-pressure liquid chromatography
Unable to display preview. Download preview PDF.
- Buters JT, Schiller CD, Chou RC. A highly sensitive tool for the assay of cytochrome P450 enzyme activity in rat, dog and man. Direct fluorescence monitoring of the deethylation of 7-ethoxy-4-trifluoromethylcoumarin. Biochem Pharmacol. 1993;46:1577–84.Google Scholar
- Chang T, Levine M, Bandiera SM, Bellward GD. Selective inhibition of rat hepatic microsomal cytochrome P-450. I. Effect of the in vivo administration of cimetidine. J Pharmacol Exp Ther. 1992;260:1441–9.Google Scholar
- Ekins S, Murray GI, Watson RD, Merle O, Hawksworth GM, Burke MD. A new substrate for the fluorescence detection of cytochrome P450 activity in individual cells. Hum Exp Toxicol. 1996;15:147.Google Scholar
- Massey TE, Devereux TR, Fouts JR. Biotransformation of xenobiotics in individual rabbit hepatocytes. Application of microspectrofluorometry. Drug Metab Dispos. 1986;14:319–24.Google Scholar