Probing enhanced cytochrome P450 2B1/2 activity in rat hepatocyte spheroids through confocal laser scanning microscopy

E. S. Tzanakakis, C. C. Hsiao, T. Matsushita, Rory P Remmel, Wei-Shou Hu

Research output: Contribution to journalArticle

27 Citations (Scopus)

Abstract

Cytochrome P450 (CYP450) enzymes are essential for xenobiotic metabolism. Although CYP450s are found in many tissues, CYP2B1/2 are primarily expressed in the rat liver. The constitutive expression in vivo of CYP2B1/2 is low but it is induced in the presence of various drugs such as phenobarbital (PB). In this study, CYP2B1/2 activity in cultured hepatocytes was assessed in situ with the introduction of a fluorogenic substrate, pentoxyresorufin. The product of 7-pentoxyresorufin-O-dealkylation (PROD), which is catalyzed specifically by CYP2B1/2, was detected using confocal laser scanning microscopy (CLSM). Primary hepatocytes cultured as monolayers on collagen-coated surfaces exhibited background PROD activity and minimal PB inducibility after 4 days in culture. In contrast, rat hepatocytes organized in compacted aggregates, or spheroids, exhibited higher levels of PROD activity and retained their ability for PB induction. The results from the CLSM analysis were verified by RT-PCR and Western immunoblotting analysis. Furthermore, CLSM in conjunction with image processing techniques and three-dimensional reconstruction revealed the localization of enhanced PROD activity in the center of spheroids. The results support the use of CLSM as a powerful tool for investigating CYP2B1/2 activity in cultured rat hepatocytes.

Original languageEnglish (US)
Pages (from-to)329-342
Number of pages14
JournalCell transplantation
Volume10
Issue number3
DOIs
StatePublished - Jan 1 2001

Fingerprint

Cytochrome P-450 CYP2B1
Dealkylation
Confocal Microscopy
Rats
Hepatocytes
Microscopic examination
Scanning
Phenobarbital
Lasers
Cytochrome P-450 Enzyme System
Collagen
Metabolism
Liver
Xenobiotics
Monolayers
Image processing
Fluorescent Dyes
Tissue
Western Blotting
pentoxyresorufin

Keywords

  • CLSM
  • Deconvolution
  • Hepatocytes
  • PROD
  • Self-assembly
  • Three-dimensional reconstruction

Cite this

Probing enhanced cytochrome P450 2B1/2 activity in rat hepatocyte spheroids through confocal laser scanning microscopy. / Tzanakakis, E. S.; Hsiao, C. C.; Matsushita, T.; Remmel, Rory P; Hu, Wei-Shou.

In: Cell transplantation, Vol. 10, No. 3, 01.01.2001, p. 329-342.

Research output: Contribution to journalArticle

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