Fluorescence-microscopic measurement of intracellular cytochrome P-450 enzyme activity (ethoxyresorufin O-de-ethylation) in unfixed liver section.

Abstract

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.