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Journal of Histochemistry and Cytochemistry, Vol. 49, 1025-1032, August 2001, Copyright © 2001, The Histochemical Society, Inc.

ARTICLE

Quantitative Cytochemical Analysis of Glucose-6-phosphate Dehydrogenase Activity in Living Isolated Hepatocytes of European Flounder for Rapid Analysis of Xenobiotic Effects

Katja Winzera, Cornelis J.F. Van Noordenb, and Angela Köhlera
a Biologische Anstalt Helgoland in der Stiftung Alfred-Wegener-Institut, Department of Ecotoxicology and Ecophysiology, Cell Biology and Pathology, Bremerhaven, Germany
b Academic Medical Center, University of Amsterdam, Department of Cell Biology and Histology, Amsterdam, The Netherlands

Correspondence to: Angela Köhler, Biologische Anstalt Helgoland in der Stiftung Alfred-Wegener-Institut, Dept. of Ecotoxicology and Ecophysiology, Cell Biology and Pathology, Am Handelshafen 12, 27570 Bremerhaven, Germany. E-mail: akoehler@meeresforschung.de

There is a great need for rapid but reliable assays to determine quantitatively effects of xenobiotics on biological systems in environmental research. Hepatocytes of European flounder are sensitive to low-dose toxic stress. Glucose-6-phosphate dehydrogenase (G6PDH) is the major source of NADPH in cells and is therefore of major importance for NADPH-dependent xenobiotic biotransformation and defense against toxic injury. These facts prompted us to develop a sensitive cytochemical method to detect G6PDH activity in living isolated flounder hepatocytes using the tetrazolium salt method. The intact plasma membrane did not appear to be a barrier for substrate, co-enzyme, and dye molecules because the intracellular enzyme reaction started immediately when incubation medium was added and could be monitored in real time per individual cell using image analysis. The reaction was effectively stopped for end point measurements by using 4% formaldehyde in 0.1 M phosphate buffer (pH 5.3). The final reaction product, formazan, was stable in hepatocytes for at least 12 days at 4C. This is the first time that a chromogenic histochemical assay is applied to living cells. This approach provides an easy tool for large-scale screening of xenobiotic metabolism and cellular stress defense. (J Histochem Cytochem 49:1025–1032, 2001)

Key Words: G6PDH, enzyme activity, tetrazolium salt, primary hepatocyte culture, histochemistry, cytochemistry, image analysis, living cells, Platichthys flesus L


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