Quantitative cytochemical analysis of glucose-6-phosphate dehydrogenase activity in living isolated hepatocytes of European flounder for rapid analysis of xenobiotic effects

There is a great need for rapid but reliable assays to determine quantitati vely 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 c ells and is therefore of major importance for NADPH-dependent xenobiotic bi otransformation and defense against toxic injury. These facts prompted us t o develop a sensitive cytochemical method to detect G6PDH activity in livin g isolated flounder hepatocytes using the tetrazolium salt method. The inta ct plasma membrane did not appear to be a barrier for substrate, co-enzyme, and dye molecules because the intracellular enzyme reaction started immedi ately when incubation medium was added and could be monitored in real time per individual cell using image analysis. The reaction was effectively stop ped for end paint measurements by using 4% formaldehyde in 0.1 M phosphate buffer (pH 5.3). The final reaction product, formazan, was stable in hepato cytes 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 s tress defense.