Cell Biology and Toxicology

, Volume 23, Issue 3, pp 143–151 | Cite as

A new technique for assaying cytochrome P450 enzyme activity in a single cell

  • Z. Taira
  • D. Yamase
  • Y. Ueda


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.


CYP1A1 metabolism microspectrofluorometry single cell 



7-ethoxyresorufin (7-ER)


cytochrome P450




fluorescein isothiocyanate


high-pressure liquid chromatography


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  1. Anderson K, Wilkinson R, Grant MH. Assessment of liver function in primary cultures of hepatocytes using diethoxy (5,6) chloromethylfluorescein and confocal laser scanning microscopy. Int J Artif Organs. 1998;21:360–4.PubMedGoogle Scholar
  2. Atkins GL, Nimmo IA. The reliability of Michaelis constants and maximum velocities estimated by using the integrated Michaelis–Menten equation. Biochem J. 1973;135:779–84.PubMedGoogle Scholar
  3. Burke MD, Orrenius S. The effect of albumin on the metabolism of ethoxyresorufin through O-deethylation and sulphate-conjugation using isolated rat hepatocytes. Biochem Pharmacol. 1978;27:1533–8.PubMedCrossRefGoogle Scholar
  4. Burke MD, Murray GI, Lees GM. Fluorescence-microscopic measurement of intracellular cytochrome P-450 enzyme activity (ethoxyresorufin O-de-ethylation) in unfixed liver section. Biochem J. 1983;212:15–24.PubMedGoogle Scholar
  5. Burke MD, Thompson S, Weaver RJ, Wolf CR, Mayer RT. Cytochrome P450 specificities of alkoxyresorufin O-dealkylation in human and rat liver. Biochem Pharmacol. 1994;48:923–36.PubMedCrossRefGoogle Scholar
  6. 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
  7. 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
  8. Donato MT, Jimenez N, Castell JV, Gomez-Lechon MJ. Fluorescence-based assays for screening nine cytochrome P450 (P450) activities in intact cell sexpressing individual human P450 enzymes. Drug Metab Dispos. 2004;32:699–706.PubMedCrossRefGoogle Scholar
  9. 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
  10. Gao F, Johnson DL, Ekins S, Janiszewski J, Kelly KG, Meyer RD, West M. Optimizing higher throughput methods to assess drug–drug interactions for CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP2D6, and CYP3A4 in vitro using a single point IC(50). J Biomol Screen. 2002;7:373–82.PubMedGoogle Scholar
  11. Ghosal A, Hapangama N, Yuan Y, Lu X, Horne D, Patrick JE, Zbaida S. Rapid determination of enzyme activities of recombinant human cytochromes P450, human liver microsomes and hepatocytes. Biopharm Drug Dispos. 2003;24:375–84.PubMedCrossRefGoogle Scholar
  12. Grafstom R, Stohs SJ. Inhibitors of 7-ethoxyresorufin and 7-ethoxycoumarin de-ethylases in rat small intestinal microsomes and cells. Drug Chem Toxicol. 1981;4:147–59.PubMedGoogle Scholar
  13. Heinonen JT, Sidhu JS, Reilly MT, Farin FM, Omiecinski CJ, Eaton DL, Kavanagh TJ. Assessment of regional cytochrome P450 activities in rat liver slices using resorufin substrates and fluorescence confocal laser cytometry. Environ Health Perspect. 1996;104:536–43.PubMedCrossRefGoogle Scholar
  14. Knodell RG, Browne DG, Gwozdz GP, Brian WR, Guengerich FP. Differential inhibition of individual human liver cytochromes P-450 by cimetidine. Gastroenterology. 1991;101:1680–91.PubMedGoogle Scholar
  15. Levine M, Law EYW, Bandiera SM, Chang TK, Bellward GD. In vivo cimetidine inhibits hepatic CYP2C6 and CYP2C11 but not CYP1A1 in adult male rats. J Pharmacol Exp Ther. 1998;284:493–9.PubMedGoogle Scholar
  16. Madsen BW, McManus ME, Woodings TL, Ilett KF. Estimation of Michaelis–Menten constants in microsomal enzyme systems by the direct linear plot and least squares regression. Xenobiotica. 1978;8:453–7.PubMedCrossRefGoogle Scholar
  17. 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
  18. Mayer RT, Netter KJ, Heubel F, Hahnemann B, Buchheister A, Mayer GK, Burke MD. 7-Alkoxyquinolines: new fluorescent substrates for cytochrome P450 monooxygenases. Biochem Pharmacol. 1990;40:1645–55.PubMedCrossRefGoogle Scholar
  19. Seglen PO. Preparation of isolated rat liver cells. Methods Cell Biol. 1976;13:29–83.PubMedCrossRefGoogle Scholar
  20. Thapliyal R, Maru GB. Inhibition of cytochrome P450 isozymes by curcumins in vitro and in vivo. Food Chem Toxicol. 2001;39:541–7.PubMedCrossRefGoogle Scholar
  21. Wagner JG. Properties of the Michaelis–Menten equation and its integrated form which are useful in pharmacokinetics. J. Pharmacokinet Biopharm. 1973;1:103–21.PubMedCrossRefGoogle Scholar
  22. White IN. A continuous fluorometric assay for cytochrome P-450-dependent mixed function oxidases using 3-cyano-7-ethoxycoumarin. Anal Biochem. 1988;172:304–10.PubMedCrossRefGoogle Scholar
  23. Wilkinson GN. Statistical estimations in enzyme kinetics. Biochem J. 1961;80:324–32.PubMedGoogle Scholar

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© Springer Science + Business Media, Inc. 2007

Authors and Affiliations

  1. 1.Faculty of Pharmaceutical SciencesTokushima Bunri UniversityTokushimaJapan

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