The conversion of the carcinogen N-hydroxy-2-fluorenylacetamide to o-amidophenols by rat liver in vitro.

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 pamidophenols, N-(7-hydroxy-2-fluorenyl)acetamide and N(5hydroxy-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 titration 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