Ethoxyresorufin: direct fluorimetric assay of a microsomal O-dealkylation which is preferentially inducible by 3-methylcholanthrene

Ethoxyresorufin (7-ethoxyphenoxazone) has been synthesized to serve as a model substrate for the simple, direct, fluorimetric assay of the hepatic microsomal O-dealkylation reaction. Liver microsomes from rat and hamster O-deethylated ethoxyresorufin to resorufin (7-hydroxyphenoxazone) in an NADPH-and oxygen-dependent reaction involving the flavoprotein. NADPH-cytochrome c reductase, and cytochrome P-450. The reaction was performed in a fluorimeter cuvette and monitored directly by recording the increase in fluorescence associated with the formation of resorufin. The reaction exhibited a relatively low apparent Km which was different in the control and induced rat (150-259 nM) and hamster (40-163 nM). Phenobarbital, but not 3-methylcholanthrene, pretreatment altered the apparent Km for ethoxyresorufin deethylation observed with rat liver microsomes, whereas both inducing agents increased the apparent Km for the reaction with hamster liver microsomes. 3-Methylcholanthrene pretreatment of the animals increased the reaction apparent Vmax 70-fold in the rat and 8-fold in the hamster, whereas phenobarbital pretreatment did not stimulate the reaction in either species. Copyright © 1974 by The American Society for Pharmacology and Experimental Therapeutics