Fluorescence-microscopic measurement of intracellular cytochrome P-450 enzyme activity (ethoxyresorufin O-de-ethylation) in unfixed liver section.
A fluorescence-microscopic method has been developed for measurement of the intracellular kinetics of the cytochrome P-450 reaction, ethoxyresorufin O-de-ethylation, in individual hepatocytes in unfixed non-frozen liver sections obtained from control or 3-methylcholanthrene-pretreated mice. De-ethylation was enhanced in the presence of salicylamide, but inhibited in the presence of alpha-naphthoflavone, A total of 21 reaction rate curves (fluorescence of the metabolite, resorufin, versus time) were constructed for different individual hepatocytes and groups of two or three hepatocytes, in a total of 12 sections from four control and eight 3-methylcholanthrene-induced mice. No two reaction curves were identical, but the curves were classified, by similarity of curve shape and fluorescence intensity, into three control categories and four 3-methylcholanthrene-induced categories. It was considered that the data represented a total of seven types of hepatocytes, which differed in their ethoxyresorufin de-ethylase reaction characteristics. Hepatocytes in the sections from 3-methylcholanthrene-pretreated mice showed higher de-ethylase activities than the hepatocytes from control mice, but the increase in activity covered a wide range of values, from 3-fold to 17-fold higher than the mean control value, suggesting that 3-methylcholanthrene did not induce ethoxyresorufin de-ethylation to the same extent in all hepatocytes.
D000818:Animals; D001571:Benzoflavones / Q000494:pharmacology; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D066298:In Vitro Techniques; D007424:Intracellular Fluid / Q000201:enzymology; D007700:Kinetics; D008099:Liver / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D008297:Male; D008748:Methylcholanthrene; D051379:Mice; D008810:Mice, Inbred C57BL; D008856:Microscopy, Fluorescence / Q000379:methods*; D010078:Oxazines / Q000378:metabolism*; D012457:Salicylamides / Q000494:pharmacology
D001571:Benzoflavones; D010078:Oxazines; D012457:Salicylamides; D008748:Methylcholanthrene; C007649:ethoxyresorufin; C011512:alpha-naphthoflavone; D003577:Cytochrome P-450 Enzyme System; C031060:salicylamide