The enzymatic reduction of the N-hydroxy derivatives of 2-acetylaminofluorene and related carcinogens by tissue preparations.

Abstract

Summary An assay was developed for the enzyme system that reduces N-hydroxy-2-acetyl-aminofluorene and related hydroxamic acids (the N-hydroxy derivatives of 7-fluoro-2-acetylaminofluorene, 4-acetylaminostilbene, 2-acetylaminophenanthrene, and 2-acetylaminonaphthalene) to their respective amides. Some amine was also formed as a consequence of deacetylation. For maximum activity unfractionated homogenates fortified with pyridine nucleotides were incubated at pH 7.8 in a nitrogen atmosphere. No requirement for flavin nucleotides was demonstrated. 2-Nitrofluorene was not reduced by liver homogenates whether or not the medium was fortified with flavin nucleotides. Liver was the most active of the tissues studied for the reduction of N-hydroxy-2-acetylaminofluorene. Rat kidney, small intestinal mucosa, adrenals, or testes had 10–20% of the activity of rat liver. Various liver tumors had activities up to 40% that of adult rat liver. Hamster and rabbit liver were several times as active in the reduction of N-hydroxy-2-acetylaminofluorene as rat liver; mouse and guinea pig liver were less active. Guinea pig and hamster liver homogenates also caused extensive deacetylation. N-Hydroxy-2-aminofluorene and 2-nitrosofluorene were readily reduced to 2-aminofluorene by liver homogenates under the assay conditions; quantitation of the enzymatic reduction required the exclusion of air, acid, and alkali during the analytical prcedures. 2-Nitrosofluorene, but not N-hydroxy-2-aminofluorene, reacted with reduced glutathione at pH 7.8 in an air or nitrogen atmosphere to yield a water soluble product. 2-Aminofluorene was obtained on incubation of this product with liver homogenate or by treatment with acid or alkali under nitrogen. The synthesis of 2-nitrosofluorene, a new compound, is described.