The Conversion of the Carcinogen N-Hydroxy-2-fluorenylacetamide to o-Amidophenols by Rat Liver in Vitro: An inducible enzymatic reaction

Abstract

Abstract The enzymatic conversion of the carcinogenic arylhydroxamic acid, N-hydroxy-2-fluorenylacetamide, to the o-amidophenols, N-(1-hydroxy-2-fluorenyl)acetamide and N-(3-hydroxy-2-fluorenyl)acetamide, referred to as the isomerization of N-hydroxy-2-fluorenylacetamide, by cell fractions of rat liver has been studied. Cell fractions prepared from the livers of normal rats were unable to perform the reaction. Isomerization was stimulated by 3-methylcholanthrene and several other polycyclic aromatic hydrocarbons administered 24 hours preceding the preparation of the cell fractions. Since dl-ethionine inhibited the stimulation of the formation of the o-amidophenols by 3-methylcholanthrene and since the inhibition was counteracted by the concurrent administration of dl-methionine, the isomerization appeared to be an induced enzymatic reaction. The isomerization proceeded optimally at a pH near 7 in phosphate buffer, Tris buffer, or deionized water and, in contrast to the microsomal p-hydroxylation of N-2-fluorenylacetamide, did not require a NADPH-generating system. N-2-Fluorenylacetamide, the substrate for the NADPH-dependent formation of the p-amidophenols, N-(7-hydroxy-2-fluorenyl)acetamide and N-(5-hydroxy-2-fluorenyl)acetamide, did not serve as a substrate for the isomerization. The isomerization was dependent on the synergistic action of two components. One of these was inducible and associated with the microsomal fraction of rat liver. The second component, localized in the soluble fraction of normal rat liver, was not dialyzable and, upon gel filtration of the soluble fraction on polyacrylamide gel, was eluted with the macromolecules excluded from the gel. Resolution of the soluble fraction on DEAE-cellulose into five subfractions and assay of each fraction indicated that the highest activity was associated with a group of proteins eluted with dilute sodium phosphate buffer at pH 4.6. The formation of the reduced metabolites, N-2-fluorenylacetamide and 2-fluorenamine, by each fraction paralleled the formation of the o-amidofluorenols. These results and the further observation that the products of the isomerization and of the reduction were present, in all fractions, in a constant proportion suggested a common intermediate for both reactions. On the basis of these data, the isomerization has been tentatively formulated as a two-step reaction. In the first step N-hydroxy-2-fluorenylacetamide is dehydroxylated to a positively charged amidonium ion by an enzyme in the soluble fraction of rat liver. The second step in which hydroxyl ions add to the electrophilic carbon atoms 1 and 3 of the resonance forms of the amidonium ion yields the o-amidofluorenols. The induced microsomal enzyme is presumed to participate in this step of the reaction. Alternatively, hydride ions may be transferred to the positively charged nitrogen of the amidonium ion to give N-2-fluorenylacetamide and, via deacetylation, 2-fluorenamine.